950R86028
FOREST RESPONSE m
QUALITY AS!
DS MANUAL
T
EXPOSURE
SYSTEMS I
AND
ITS
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EPA ERL-Corvallis Library
000064
QUALITY ASSURANCE METHODS MANUAL
FOR
EXPOSURE SYSTEMS AND PHYSIOLOGICAL MEASUREMENTS
Lance S. Evans
Laboratory of Plant Morphogenesis
Manhattan College
The Bronx, NY 10471
and
Philip Dougherty
School of Forest Resources
University of Georgia
Athens, GA 30602
June 1986
library
U.S. Environmental Protection Agvnqf
National Hoaltti end Environmental
Bllocts Research Laboratory
300 S.Y7. 35th Streoi
Corvallis, Oregon 97339
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TABLE OF CONTENTS
1. Introduction 3 . . . 0 • . June 1986 1
1.1. Exposure Systems 1
1.2. Physiological Measurements 2
1.3. References 3
2. Protocol Narratives for Exposure
Systems 8 . . . 0 . . June 1986 4
2.1. Wet Deposition - Automatic Rainfall Exclusion with
Regular Additions 4
2.2. Dry Deposition - Open-Air Release (Chamberless)
Systems 5
2.3. Wet Deposition - Automatic Rainfall Exclusions with
Automatic Additions 8
2.4. Dry Deposition - Open-Top Chambers . 8
2.5. Controlled-Environment Facilities 9
2.6. Greenhouse Facilities 9
2.7. References 10
3. Protocol Narratives for Pollutant
Delivery and Monitoring Systems ... 8 . . . 0 . . June 1986 12
3.1. Wet Deposition 12
3.2. Dry Deposition 13
3.3. References 19
4. Standard Operating Procedure for
Measurement of Net Carbon Exchange. .11 . . . 0 . . June 1986 20
4.1. Scope and Purpose 20
4.2. Materials and Supplies 20
4.3. Procedures 21
4.4. Preventive Maintenance 25
4.5. Calibration Procedures 26
4.6. Calculations/Units 27
4.7. Error Allowance and Data Quality 29
4.8. References 30
5. Standard Operating Procedure for
Plant Water Relations Measurements 10 . . . 0 . . June 1986 31
5.1. Scope and Purpose 31
5.2. Materials and Supplies 31
5.3. Procedures 32
5.4. Preventive Maintenance 37
5.5. Calibration Procedures 37
5.6. Calculations/Units 38
5.7. Error Allowance and Data Quality 39
5.8. References 40
ii
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CONTENTS (cont'd)
6. Standard Operating Procedure for
Measurement of Seedling Leaf Area. . . 7 . . . 0. .. June 1986 41
6.1. Scope and Purpose 41
6.2. Materials and Supplies 41
6.3. Procedures 41
6.4. Preventive Maintenance . 46
6.5. Calibration Procedures . 46
6.6. Calculations/Units 47
6.7. Error Allowance and Data Quality 47
6.8. References 48
7. Standard Operating Procedure for
Seedling Growth Measurements 6 . . . 0. . June 1986 49
7.1. Scope and Purpose 49
7.2. Materials and Supplies • 49
7.3. Procedures • 49
7.4. Preventive Maintenance 51
7.5. Calibration Procedures 51
7.6. Calculations/Units 52
7.7. Error Allowance and Data Quality 52
7.8. References ..... 53
8. Standard Operating Procedure for
Determination of Foliar Injury to
Seedlings and Saplings 2 . . . 0. . June 1986 55
8.1. Scope and Purpose 55
8.2. Materials and Supplies 55
8.3. Procedures , 55
8.4. Preventive Maintenance 56
8.5. Calibration Procedures 56
8.6. Calculations/Units 56
8.7. Error Allowance and Data Quality 56
8.8. References 56
9. Standard Operating Procedure for
Mycorrhizal Assessments 3 . . . 0. . June 1986 57
9.1. Scope and Purpose 57
9.2. Materials and Supplies 57
9.3. Procedures 57
9.4. Preventive Maintenance 58
9.5. Calibration Procedures ..... 58
9.6. Calculations/Units 58
9.7. Error Allowance and Data Quality 58
9.8. References 59
iii
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CONTENTS (cont'd)
Section
Pages Revision Date Page
References
A .... 0 . .June 1986 60
Appendices
33
0 . .June 1986 64
A. List of Attendees
65
B. Table of Measurements, SOP References, and Precision
Levels Required for Measures of Net Carbon Exchange,
Water Vapor Exchange, Xylem Pressure Potential, and the
Required Associated Environment and Plant Variables. ... 67
C. LI-6000 Portable Photosynthesis System - Service included
with 6000 CAL Factory Calibration 69
D. Computer Codes and Reporting Symbols for Certain Measured
Variables 71
E. LI-1600 Steady State Porometer 1600CAL Factory Calibration
Description 72
F. National Crop Loss Assessment Network Quality Assurance Plan
for Biological Measurements. ............... 73
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PREFACE
The following Forest Response Program (FRP) Methods Manual for
Exposure Systems and Physiological Measurements was developed in response
to the Environmental Protection Agency's (EPA) requirements for Quality
Assurance (QA) in environmental research. The Exposure Systems workshop
which provided the basis for this document was held on March 11-12, 1986
at Raleigh, NC. The workshop was attended by 24 individuals consisting of
Principal Investigators and Program Administrators (see Appendix A). The
Exposure Systems workshop is one of four workshops conducted by the EPA to
develop Quality Assurance Methods Manuals for the Forest Response Program.
The workshops and ensuing reviews of the Methods Manuals were designed to
allow scientists input in the selection of standard research methods and
the development of QA procedures for these methods. Drs. Lance Evans and
Phil Dougherty provided workshop leadership and compiled this methods
manual from the comments and concerns voiced at the workshop, and other
sources.
The protocols and Standard Operating Procedures (SOPs) presented in
these methods manuals represent a consensus on the "best" technique for
operating a specified system of equipment or measuring a specified
variable given the various objectives of the Forest Response Program.
"Best" is defined for this manual according to the following criteria:
1) the quality or soundness of the protocol or SOP;
2) the availability of staff and facilities to adhere to the
protocol or SOP; and
3) the feasibility of assuring data quality through tests for
accuracy, precision, and consistency among sites.
The protocols and SOPs written in this first edition of the manuals are
general in nature and encompass most existing research plans. As the
program progresses, the protocols and procedures will be revised, and the
manuals will be expanded to include additional protocols and SOPs.
The purpose of the manual is (1) to provide standardization of
research methods and design to allow for the synthesis and integration of
results for assessment purposes; (2) to provide standard quality
assurance/quality control techniques within standardized protocols and
SOPs to allow for the assessment and documentation of data quality; and
(3) to prevent duplication of documentation efforts among investigators
using common techniques. Appropriate standardization of research methods
which contribute data to a centralized data base is critical to the
synthesis/integration/assessment effort for the Forest Response Program.
Without comparability among sites, an overall assessment of research
results would be impossible. The National Forest Response Program
Research Plan provides details on the integration and assessment effort.
Standard quality assurance activities, providing guidelines for the
minimum amount of activity required to participate in the integrated
research program, ensure the ability of investigators and Program
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Administrators to assess data quality as data are produced. This
knowledge directly influences the level of confidence for assessment
decisions, which is determined from the quality of the individual parts.
Other aspects of the quality assurance program (research plan preparation,
auditing, and sample exchanges between sites) contribute to the process to
ensure that data are of known and sufficient quality to meet the program's
objectives.
Finally, these methods manuals will serve the investigators in simply
reducing the amount of documentation required for the assessment of data
quality. Many of the techniques employed by investigators examining
similar hypotheses are nearly identical. The manuals identify and
document these similarities and can be referenced by the investigators in
their research plans. Much of the information required by the QA program
is included in these manuals; however, instances occur when generalities
should be clarified in individual plans. For example, "sufficient
training to operate the required equipment" as might be found in a manual
must be specified for projects and facilities in the individual research
plan since large differences may exist between sites and projects.
All investigators with research projects funded through the Forest
Response Program Research Cooperatives will be required to adhere to the
protocols and the SOPs as described in these manuals. Where necessary,
investigators can deviate from the manual, by providing (1) a
justification for the deviation; (2) a full explanation of the alternative
protocol or procedure with QA activities clearly described; and (3) an
assessment of the impact of the deviation on data quality, by comparing
the alternative protocol or procedure with the original. This will create
an additional documentation burden for the investigator and an assessment
burden for administrators, and therefore, should be used only when
absolutely necessary.
The Forest Response Program Quality Assurance staff would like to
thank the workshop leaders, Dr. Evans and Dr. Dougherty, and the
participants for their technical efforts in developing and refining the
material in this manual, and the Acid Deposition Program staff for
organizing and supporting the effort. Ue appreciate the cooperation and
patience of all the investigators during the slow process of developing
the QA program, including this manual. We look forward to the ensuing
years of quality research and an integration and assessment effort worthy
of that quality.
NOTE: The use of brand names in this manual is not an endorsement of a
particular product but a guide to the type of equipment required to
perform a particular measurement.
vi
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1. INTRODUCTION
The recently established Forest Research Cooperatives are charged
with the task of understanding the nature of forest declines, if forest
declines are occurring, as well as making an economic assessment of
effects at some date in the future. This is an ambitious task and will
only be accomplished if effective and efficient planning occurs before
experimentation begins.
The purpose of an assessment is to present the best answers or
predictions in response to policy questions with a clear discussion of all
uncertainties (Moskowitz et al., 1985). For any assessment activity, only
limited data will be available and certain inferences will be made from
available data bases to predicted regional or national impacts. Of
course, any prediction carries uncertainties. Most accurate predictions
can be made when the magnitude of each uncertainty can be characterized
or, at least, be placed within certain limits.
1.1. EXPOSURE SYSTEMS
This document is constructed with the protocols as outlined in the
recent International Workshop on Standardization of Pollutant Exposure
Systems and Protocols held in Corvallis, OR, in January 1986, as a
foundation (Table 1) (Hogsett et al., 1986). Participants at the workshop
recognized that the "ideal" system for multi-year growth studies with both
seasonal and year-round exposure to different pollutant types would be
structureless, i.e., not introducing artifacts of growth with hardware and
shelters. Participants recognized that experimentation may involve
certain tradeoffs. One tradeoff is the need to exclude ambient pollutants
(wet and dry deposition), and a means to accomplish this is a chamber or
enclosure. The automated exclusion systems, in use at some acid-rain
deposition sites, represents an attempt to minimize the artifacts except
during the ambient event (i.e., rain event). The workshop participants
recognized that, since researchers lacked an "ideal" or structureless
exposure system that was well-characterized for tree growth and pollutant
delivery, there vas value in employing some more adequately characterized
exposure systems. Thus, this allows the opportunity of cross-
comparability between research sites with some systems, but avoids the
undesirable introduction of consistent bias into the programs. The
workshop recognized the utility of a variety of exposure systems for
multi-year growth studies, including both chambered and non-chambered type
systems.
Some experimentation may need to take place under conditions in which
mechanisms of response, initial stages of screening sensitive plant
species and/or genetic variants, or other considerations such as
accessibility to remote areas or characteristics of the environment may
make certain exposure systems inappropriate. Under such conditions it may
be necessary to conduct wet deposition studies under a permanent exclusion
cover or in a controlled environment facility. In a likewise manner, dry
deposition studies may need to be conducted in controlled environment
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conditions. It is acknowledged that none of the exposure systems have
been adequately tested for performance with forest trees. Regardless of.
system(s) used, tree growth and physiology should be accurately
characterized so that pollutant effects can be separated from exposure
system effects. Nevertheless, it is recognized that the more the
experimental environment differs from the natural environment, the more
problematic inferences become when experimental results are used for
regional or national assessments.
Table 1. Recommended exposure systems for combinations of dry and wet
deposition. (The order does not imply prioritization.) Table
is printed from Table VI-6 of the report of the International
Workshop on Standardization of Pollutant Exposure Systems and
Protocols, Corvallis, OR, January 1986. (Hogsett et al., 1986)
Multi-Year Growth/Physiology Studies
Wet Depos
Dry Depos
Wet Depos
Dry Depos
Wet Depos
tion — Automated or manual exclusion/scheduled addition
tion — Chamberless exposure system
tion — Automated or manual exclusion/scheduled addition
tion — Open-top chamber technology
tion — Permanent exclusion/scheduled addition
- Open-top chamber technology
Dry Deposition -
Short-Term Growth/Physiology Studies
Same as multi-year plus indoor chambers
1.2. PHYSIOLOGICAL MEASUREMENTS
The purpose of the physiological measurement section is to define
acceptable standards for measuring the key physiological processes that
are of interest to the Forest Response Program. The measurements
addressed in this manual are those made at the whole plant or organ level
and not those made at the subcellular or biochemical level.
Exact methods, numbers of plants to be sampled, and frequency of
sampling cannot be specified for all studies because they will be a
function of study objectives. The accuracy and precision of each
measurement has been specified where possible for each of the
physiological measurements considered in this document. This will require
that equipment with suitable resolution and stability be used. Otherwise,
specific equipment recommendation has been avoided.
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In this section of the manual it has been proposed that standard
units and recording formats be accepted for each physiological process
measured. Adherence to these guidelines vill make data management and
data analysis easier.
1.3. REFERENCES
Hogsett, W. E. , D. P. Ormrod, D. Olszyk, G. E. Taylor, Jr., and
D. T. Tingey. 1986. Air Pollution Exposure Systems and Experimental
Protocols: A Review and Evaluation of Performance. (In preparation)
Moskovitz, P. D., U. H. Medeiros, N. L. Oden, H. C. Thode, Jr.,
E. A. Coveney, J. S. Jacobson, R. E. Rosenthal, L. S. Evans,
K. F. Lewin, F. L. Allen. 1985. Effects of Acid Deposition on
Agricultural Production, BNL51889, Brookhaven National Laboratory,
(Jpton, New York.
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2. PROTOCOL NARRATIVES FOR EXPOSURE SYSTEMS
2.1. WET DEPOSITION - AUTOMATIC RAINFALL EXCLUSION VITH REGULAR ADDITIONS
Automatic rainfall exclusion systems equipped to apply simulated
precipitation on a regular schedule were recommended for wet deposition
studies by the International Workshop on Standardization of Pollutant
Exposure Systems and Protocols held at Corvallis, Oregon, during January
1986 (Hogsett et al., 1986). The concept behind the use of such
facilities centers around the need to exclude all ambient wet deposition
and apply deposition of known chemistry, quantity, etc. as well as the
need to change the microclimate of the environment only minimally so that
the experimental environment most nearly mimics the natural environment.
These systems have been constructed at several sites in North America
based upon the facility at Brookhaven National Laboratory (Levin and
Evans, 1985). These facilities do not exclude dry deposition.
2.1.1. SYSTEM CHARACTERISTICS
° These facilities should be made of construction materials such
that no structural damage should occur at wind speeds of up to
23 m/s at low elevations.
° At high elevations (above 1000 m) the structures should be able to
withstand wind speeds of up to 41 m/sec.
0 The exclusion system should exclude ambient precipitation within
three minutes after sensors experience the first droplet.
0 The shelter should be moved off the experimental area within five
minutes after precipitation stops.
" A real-time recorder system must monitor when the shelter is over
the experimental area.
° All records of time are to be reported in standard military time.
0 No surface water should enter the exclusion area.
0 The simulated rain distribution system should be a permanent part
of the shelters and should not be in the experimental area unless
the shelter is over the area.
° The covering of the shelters should not transmit less than 75% of
ambient light - 90% is recommended level, with transmission to 75%
of ambient in the PAR spectrum region. If the light intensity
decreases below this level, the cover should be cleaned or
changed.
0 The temperature of the treatment area should not differ by more
than 3°C from ambient.
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° The relative humidity of the air of the treatment area should not
differ by more than 10% from ambient.
° All instruments to determine and record temperature and relative
humidity should be calibrated monthly during experimentation.
° Climatological data of ambient conditions should be recorded
continuously. Wind speed within the shelter during simulated
precipitation events must be below 1 m/sec so that the pattern is
not significantly affected.
2.2. DRY DEPOSITION - OPEN-AIR RELEASE (CHAHBERLESS) SYSTEMS
Chamberless exposure systems were recommended by the International
Workshop (Hogsett et al.,) for dry deposition studies attempting to mimic
natural conditions. The chief advantage of open air release
(structureless exposure) systems is that they dispense gaseous air
pollutants vith a minimum of perturbations of the natural environment and,
as a result, experimental results from such experiments are most
applicable for assessment purposes. One limitation of these systems is
the lack of an imposed "no pollution" control unless the experimental
field is located in such an environment or a gas without pollutants is
administered. Many systems have been developed (de Cormis et al., 1975;
Lee and Levis, 1978; Heitschmidt et al., 1980; Muller et al., 1979; Miller
et al., 1980; Greenwood et al., 1982; McLeod et al., 1985; Mooi and van
der Zalm, 1985a, 1985b). The performance records of the systems described
by Greenwood et al. (1982), McLeod et al. (1985), and Mooi and van der
Zalm (1985a, 1985b) are probably the most complete and were used as guides
for this report.
The configuration of pipes used by Mooi and van der Zalm (1985a,
1985b) is a new development. They used 16 pipe segments in a circular
array in such a way that each pipe could be operated independently. In
this manner, each of the 16 pipe segments would supply test gases to only
22 (360/16) degrees within the circle's area. Only pipes up-wind of the
experimental area are activated at any one time. The concentration of SO^
in the experimental area is maintained by computer. Results of tests
during the latter portion of 1984 show that the average concentration in
the center of the experimental field could be maintained at 200 yg/m SOj.
The deviation of this average was about 15% (average of 10 consecutive
measurements within a period of 16 minutes). Higher peaks were extremely
rare. Distribution of S0£ gas over the whole experimental field (diameter
10 m) had a deviation of about 20%. This level of control should be
attempted in all experiments.
To operate this system, the following environmental data must be
monitored continuously within the experimental area:
- wind speed in m/s
- wind direction
- temperature in degrees Celsius
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- pollutant concentration at each sample point (at least three
sampled sites at two heights per test plot)
- background concentrations of pollutant.
The QA documentation needs a description of how the automated system
operates. In case of perceived alarms in the systems, the following
problems will call for particular actions. The alarms and actions are:
1. If pollutant concentration is too high (202 above set point),
injection will be reduced markedly.
2. The automatic system should be checked periodically.
3. All instruments should be calibrated according to schedules that
follow.
4. An instrument should be dedicated to surveillance of one system
on a constant basis.
5. All gas distribution lines should be monitored frequently to
assure accurate flow rates.
6. QA checks should be made by adding pollutants and monitoring how
the automated system responds.
The characteristics of performance for 0^, SC^, and NO^ analyzers are
shown in Table 2. For open field exposure conditions, a low detectable
limit, high degree of specificity, high stability, and rapid response time
are very important. Such characteristics are less important when chamber
conditions are used, because the pollutant concentrations can be
controlled more accurately. For open air situations, the following
conditions should be monitored during the experimental period.
1. Atmospheric Chemistry
(A) Gaseous Pollutants
(1) Distribution Patterns
(i) Horizontal — continuous monitoring at many points
in a grid over plot (minimum of three points)
(ii) Vertical — systematic monitoring at two heights in
each plot (minimum of two points)
(2) Temporal Patterns — continuous at many points
(B) Non-Pollutant Chemicals
(1) Water Vapor (Humidity) — continuous at one point in
ambient air
(2) CO2 — not applicable
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Tsble 2. Perfomanca characteristic! of aany gaseous polio tint analysara.
TYPICAL OZONE/OXIDANT ANALYZERS
Analyser
Parame ter
Colorloetrlc
Gas-Phase
Cheml luminescent
Gas-Solid
Chemlluminescent
nv Photonetrie
Electrochemical
Lower Detect-
10-20 ppb
< 1-10 ppb
< 1-10 ppb
< 1-10 ppb
10-20 ppa
able Llnit
Specificity*
Ion
high
high
high
low
Stability15
low
high
high
high
low
Response Tine
< 3 nln
< 1 Din
< 1 Din
< 1 oln
< 5 nln
Working Range
up to 10 ppo
up to 2 ppo
up to 2 ppm
up to 10 ppm
up to 10 ppm
Cost (dollars)
3-6 K
3-6 K
> 6 K
3-6 K
1-3 K
TYPICAL SOj ANALYZERS
Analyser
Parameter
Conducti-
ve trie
Voltame trie
Aoperooetrie
Colorloetrlc
Flame
Photometric
2nd
Derivative UV
Spectroaetrlc
Pulsed DV
Fluorescent
Lower Detect-
10-20 ppb
10-20 ppb
10-20 ppb
10-20 ppb
< 1-10 ppb
10-20 ppb
10-20 ppb
able Limit
Specificity*
siodera te
aoderate
Boderate
high
ooderate
high
ooderate
Stability11
low
low
high
low
high
high
high
Response Time
< 5 mln
< 5 Bin
< 5 Bin
< 5 Bin
< 1 Bin
< 5 mln
< 5 Bin
Working Range
up to
up te
up to 2 ppm
up to 4 ppm
up to
up to 2 ppo
up to 3 ppm
10 ppm
10 ppa
10 ppm
Cost (dollars)
1-3 K
1-3 K
3-6 K
3-6 K
> t K
< 6 K
> 6 K
TYPICAL HO, ANALYZERS
Analyser
Parameter
Toltaaetrie
Amperometrlc
Colorloetrlc
Chemlluminescent
Lower Detee t-
10-20 ppb
10-20 ppb
10-20 ppb
< 1-10 ppb
able Limits
Specificity*
low
low
high
low
Stability11
low
low
low
low
Response Tioe
< 5 Bin
< 3 Bin
> 3 oin
< 1 Bin
Working Range
up to 10 ppm
up to 12 ppm
up to 2 ppm
up to 10 ppm
Cost (dollars)
1-3 K
3-6 K
3-6 K
(61
'Specificity: high — < 10% error from speclea commonly encountered In anblent air; ooderate — scrubber required
to ellninate Interferences; low — scrubber may not eliminate Interferences under all conditlona, and/or data
corrections required based an concurrent neasurements.
^stability! high -- meet EPA Reference and Equivalent Method specifications; ooderate •• oay be operated
without significant drift for 1-2 days; low — requlrea dally sero/span adjustment.
Sourcei fiurnann and Rehae (1978)*
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2. Physical Properties of the Experimental Area
A meteorological station should be established to monitor the
ambient environment. This station should be constructed and
operated according to US EPA guidelines.
(A) Irradiance (Quantum Level at 400-700 nm)
(1) Distribution — not applicable
(2) Temporal Patterns — Continuous monitoring at one point
in ambient air
(B) Air Temperature — Continuous at one point in ambient air
(C) Air Movement — Continuous wind speed measurements are
necessary as stated above
(D) Soil Temperature — Periodically at one point in ambient plot
area
2.3. WET DEPOSITION - AUTOMATIC RAINFALL EXCLUSION VITB AUTOHATIC
ADDITIONS
The International Workshop (Hogsett et al., 1986) recommended
(1) automatic rainfall exclusion with automatic simulant addition and
(2) permanent exclusion cover systems for particular circumstances. The
first system, automatic rainfall exclusion with automatic simulant
addition, vas used in a study by Shriner et al. (1985). The advantages
and disadvantages of this method have been reviewed by Hogsett et al.,
(1986). These protocols are subject to the same requirements as described
above for the first system described for wet deposition. Fev additional
changes in this document are needed to accommodate this protocol compared
with the system which uses scheduled simulant additions. Documentation of
volumes of simulated rain in real time as well as volumes of rain under
ambient conditions must be recorded in automated simulant addition
systems.
A permanent exclusion cover system may be used under certain
circumstances. Vhen a permanent cover is used, data to characterize the
environment must be in place. Such environmental factors as air and soil
temperature, relative humidity, light intensity (PAR), wind direction and
velocity, and calculations of evapotranspiration must be made continuously
at a minimum of one point pEi set of identical chambers, but aore may be
necessary depending upon variability*
2.4. DRY DEPOSITION - OPEN-TOP CHAMBERS
The International Workshop (Hogsett et al., 1986) recommended the use
of open-top chambers, common to the National Crop Loss Assessment Network
(NCLAN). Other research groups have used them as veil. The technology is
well developed and standardized (Heagle et al., 1973; Mandl et al., 1973;
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Rats et al., 1974; Olszyk et al., 1980). In terms of quality control, all
mechanical parts of such equipment must be maintained. The plastic should
be cleaned frequently to maintain the highest quality light transmission.
The plastic should be replaced when light transmission into the chamber is
below 90X of a new panel. Chambers should be operated to provide at least
three air changes per minute. One typical chamber will be monitored
continuously for light transmission, air velocity, relative humidity, and
pollutant concentrations. In this typical chamber, pollutant
concentrations will be monitored continuously at two locations at canopy
height. All filters will be checked daily during operation.
As a minimum, one gas inlet pipe for pollutant monitoring will be
located in each chamber. These sample points should be located near the
central portion of each chamber at canopy height. Automatic recordings of
real-time data should be made available at all times. These chambers are
subject to the same "alarms" as for the open-air protocol listed above.
It must be recognized that air temperature and relative humidity are
important determinants of plant growth and that, as with other systems, a
restriction of air flow in these systems would result in elevated air
temperature and relative humidity. Air temperature, light intensity, and
relative humidity should be monitored continuously at the center of the
chamber, and at canopy height in one chamber. Air flow rates must be
maintained to the extent that the temperature in the chamber never exceeds
3°C above ambient conditions. If this limit is exceeded, the system
should be reevaluated as soon as possible,
2.5. CONTROLLED-ENVIRONMENT FACILITIES
Many recent publications have focused on methods, timing, and data
reporting, including units of expression of environmental condition for
controlled-environment facilities (Berry et al., 1977; McFarlane, 1981;
Spomer, 1981; Krizek and McFarlane, 1983). The guidelines of Krizek and
McFarlane (1983) should be followed. These facilities are subject to the
same types of monitoring and instrumentation quality assurance as all
other types of facilities. The environments in such systems should be
described in terms of temperature, relative humidity, light intensity in
the PAR portion of the spectrum, and pollutant exposures. Pollutant
exposures should not vary by more than 10% of set point and pollutant
concentrations in all chambers should be monitored continuously on a time
sharing basis. A strip chart recorder should be operated continuously
with this monitor. Temperature, light intensity in PAR, and relative
humidity should be monitored during fumigations in a sufficient number of
chambers to guarantee that there are no significant differences among
chambers.
2.6. GREENHOUSE FACILITIES
Quality assurance for greenhouse facilities is different from other
previously described facilities. Such facilities do not provide the
careful controlled environments of growth chambers or the ambient
conditions of open field conditions. For quality assurance purposes,
temperature, relative humidity, and light intensity in the PAR should be
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monitored continuously. Periodic checks on background air pollutant
concentrations, dust, etc. should be made. Temperatures are very
important in experiments with forest species. An understanding of
temperatures under ambient forest conditions may preclude a specific
experimental approach. For example, temperatures in greenhouses should
not exceed 90°F for studies with red spruce since this species rarely
experiences this temperature in the forest. In many greenhouse facilities
the only economical approach to cooling is provided by shading of various
types. One unfortunate effect of shading is plant etiolation. It is
specified that shading should not exceed 40% of PAR. It should be noted
that the growing environment of controlled conditions can have profound
effects on plant anatomy, physiology, and pollutant responses. In
controlled environment facilities and greenhouse experiments, the most
acceptable measure of pollutant concentration of exposure is the
concentration measured in the exhaust line. The exposure containers
should not change pollutant chemistry.
2.7. REFERENCES
Berry, V.L., P.A. Hammer, R.H. Hodgson, D.R. Krizek, R.U. Langhans, J.C.
McFarlane, D.P. Ormrod, H.A. Poole, and T.W. Tibbitts. 1977. Revised
guidelines for reporting studies in controlled environment chambers.
Hort. Science 12:309-310.
Burmann, F.J. and K.A. Rehme. 1978. Instrumentation, pp. 2-1 to 2-24,
In: Handbook of Methodology for the Assessment of Air Pollution
Effects on Vegetation, W.V. Heck, S.V. Krupa, and S.N. Linzon, eds.,
Air Pollution Control Association, Pittsburgh.
de Cormis, L., J. Bonte, and A. Tisne. 1975. Technique experimentale
permettant l'etude de 1'incidence sur la vegetation d'une pollution
par le dioxyde de soufre appliquee en permanence et a dose
subnecrotique. Pollut. Atmos. 17:103-107.
Greenwood, P., A. Breenhalgh, C. Baker, and M. Unsworth. 1982. A
computer-controlled system for exposing field crops to gaseous air
pollutants. Atmos. Environ. 16:2261-2266.
Heagle, A.S., R.B. Philbeck, and W.V. Heck. 1973. An open-top chamber to
assess the impact of air pollution on plants. J. Environ. Qual.
2:365-368.
Heitschmidt, R.K., W.K. Laurenroth, and J.L. Dodd. 1978. Effects of
controlled levels of sulphur dioxide on Western Wheatgrass in a
southeastern Montana grassland. J. Appl. Ecol. 15:859-868.
Hogsett, W.E., D.P. Ormrod, D. Olszyk, G.E. Taylor, Jr., and D. T. Tingey.
1986. Air Pollution Exposure Systems and Experimental Protocols: A
Review and Evaluation of Performance. (In preparation)
Kats, G., C.R. Thompson, and W.C. Kuby. 1974. Improved ventilation of
open top greenhouses. J.Air Pollut. Contr. Assoc. 26:1089.
10
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Krizek, D.T. and J.C. McFarlane. 1983. Controlled-environment
guidelines. Hort. Science 18:662-664.
Lee, J.J. and R.A. Lewis. 1978. Zonal air pollution system: Design and
performance. Ecological Research Series EPA 600/3-78-021, pp. 322-
344, U.S. Environmental Protection Agency, Corvallis, OR.
Levin, K.F. and L.S. Evans. 1985. Design and operation of an
experimental system to determine the effects of rainfall acidity on
vegetation. BNL 34649, Brookhaven National Laboratory, Upton, NY.
Mandl, R.H., L.H. Weinstein, D.C. McCune, and M. Keveny. 1973. A
cylindrical open top field chamber for exposure of plants to air
pollutants in the field. J. Environ. Qual. 2:371—376.
McFarlane, J.C. 1981. Measurement and reporting guidelines for plant
growth chamber environments. Plant Science Bui. 27(2):9—11.
McLeod, J.A.R., J.E. Fackrell, and K. Alexander. 1985. Open-air
fumigation of field crops: Criteria and design for a new
experimental system. Atmos. Environ. 19:1639-1649.
Miller, J.E., D.G. Sprugel, R.N. Muller, H.J. Smith, and P.B. Xerikos.
1980. Open-air fumigation system for investigating sulfur dioxide
effects on crops. Phytopathology 70:1124-1128.
Mooi, I.J. and A.J.A. van der Zalm. 1985a. Research on the effects of
higher than ambient concentrations of SO2 and N0„ on vegetation under
semi-natural conditions: The developing and testing of a field
fumigation system; process description. First Interim Report,
January-December 1983.
Mooi, I.J. and A.J.A. van der Zalm. 1985b. Research on the effects of
higher than ambient concentrations of SO2 and N0„ in vegetation under
semi-natural conditions: The developing and testing of a field
fumigation system; Execution. Interim Report, January-December,
1984.
Muller, R.N., J.E. Miller, and D.G. Sprugel. 1979. Photosynthetic
response of field-grown soybeans to fumigations with sulphur dioxide.
J. Appl. Ecol. 16:567-576.
Olszyk, D.M., T.M. Tibbitts, and W.M. Hertsberg. 1980. Environment in
open-top field chambers for air pollution studies. J. Environ. Qual.
9:610-615.
Shriner, D.S., J.W. Johnston, Jr., G.E. Taylor, Jr., R.J. Luxmoore, R.K.
McConathy, S.B. McLaughlin, A.S. Heagle, R.J. Norby, B.K. Takemoto,
D.T. DuBay, C.H. Abner, D.D. Richter. 1985. Acidic deposition:
Effects on agricultural crops. Final Report to the Electric Power
Research Institute (Project 1908-2).
Spomer, L.A. 1981. Guidelines for measuring and reporting environmental
factors in growth chambers. Agron. J. 73:376-378.
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3. PROTOCOL NARRATIVES FOR POLLUTANT DELIVERY AND MONITORING SYSTEMS
3.1. WET DEPOSITION
3.1.1. GUIDELINES
The following items should be used as guidelines for mixing,
delivering, and monitoring wet depositions.
0 Water to be used for simulated wet deposition should not be held in
storage that influences its quality. Some justification is
required to show that the container or the holding duration do not
influence water quality.
0 Only deionized or distilled water below 5 megohm conductivity
should be used to make simulated precipitation. If the
conductivity is above this limit, investigate and correct. The
water used should be monitored for conductivity continuously.
0 When simulants are being applied, the pH of the solution must be
monitored continuously by at least one calibrated pH meter. Two pH
meters are recommended. Differences between pH readings should be
investigated and corrected. Calibrations should be made with a
solution of relatively low specific conductivity.
0 The distribution patterns and volumes should not vary by more than
10% of mean delivery volume per unit area within each plot among
plots of the same treatment. Checks of patterns and volumes should
be done once monthly, four areas per chamber, when the shelters are
not over the treatment area.
0 All nozzles from all plots should be removed and cleaned every two
weeks of operation. Nozzles should be placed back into the system
in a random manner.
0 During each treatment application, grab samples from several
nozzles should be monitored for pH and conductivity three times
during life of stock solution.
° The mixing and distribution systems should remain closed, except to
flush unwanted water, at all times so contamination from the
environment cannot occur.
0 Each pH meter must be calibrated with standardized buffers covering
the pH range of test solutions prior to each day's treatments and
the calibrations recorded in addition to usual testing.
° Two times per year, predetermined random grab samples of each
acidity level should be obtained from nozzles and sent to an
independent laboratory for analysis.
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0 The pH levels of the simulated vet deposition should be established
by the PI after consulting with the particular Research
Cooperative. The Forest Response Program should recommend pH
levels to be used which are based upon assessment needs. The PI
should have some recommended levels. Of prime importance is the
number of treatments and the number of replicate plots of each
treatment. The number of treatments is primarily determined by the
method of statistical analysis used for assessment purposes. The
number of plots per treatment should be maximized. At least 4
plots per treatment is required. At least 10 plots per treatment
is strongly recommended.
3.2. DRY DEPOSITION
3.2.1. GUIDELINES
The generation of gaseous pollutants has been discussed briefly in a
previous section. These systems are described in Table 2 (section 2.2).
Each method has its benefits and limitations. The pollutants and the
treatment regimes for each experiment can only be established by the PI
(Pis) after consulting with the particular Cooperative Directors. Only
treatments which reflect real-world conditions should be used in
experiments to mimic natural conditions. Experimental exposures should be
based upon exposure conditions that actually occur in nature. Therefore,
treatments that implement (1) rapid changes in gas concentrations, (2)
exposures to two gas pollutants that co-occur very rarely, or
(3) unusually high gas concentrations should be avoided if possible.
Under some circumstances, treatment levels outside these limits might be
necessary to provide a more neaningfull dose-response function. Of prime
importance is the number of treatments and the number of- replicate plots
of each treatment. The number of treatments is primarily determined by
the method of statistical analysis. The number of plots per treatment
should be maximized.
The assurance of quality from all tests with gaseous pollutants
depends upon the accuracy of the analyzer/controller systems. The
following narrative follows the general format of that used by the
National Crop Loss Assessment Network (NCLAN).
When a large number of treatments are administered, time-sharing of
air pollution monitors is a frequent occurrence. The sampling time of
each area or unit should be 3 to h times longer than the minimum response
time {see Table 2) to insure adequate monitoring. The International
Workshop (Hogsett et al., 1966) encouraged a sampling frequency sufficient
to provide characterization of an hourly average concentration. This
situation is particularly relevant when monitoring SO2 at temperatures
below 20°C with relative humidities above 80£.
Under most gaseous exposure regimes it is necessary to mimic, as
closely as possible, temporal variations that are normally experienced in
polluted areas. For ozone exposures this would constitute relatively low
concentrations between 1900 hrs and 0B00 hrs daily. Between 0800 to
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1900 hr daily, the 0^ concentration should increase, plateau, and
eventually decrease. The rate of increase and decrease as well as the
duration of the plateau should follow ambient as much as possible. This
protocol should be used in field applications and under most controlled-
environment applications.
Under some circumstances that use controlled-environment
applications, the protocol of a gradual increase and gradual decrease in
gas concentration may be very impractical in terms of time, equipment, and
financial support. Under these circumstances, a rapid increase and rapid
decrease in gas concentration (i.e. square wave) may be used. Such
protocols can be easily standardized and monitored, and the results are
conveniently expressed. This latter protocol may be used in experiments
of a preliminary nature such as screening plant species or various genetic
isolines of a particular species, as well as determining, on a preliminary
basis, dose-response relationships between pollutants and responses. In
addition, such exposure protocols may be used for pollutants in which air
quality data are poor. This protocol should not be used for assessment
purposes.
Seventy-five percent of the total possible observations during a
sampling period must be present to meet completeness requirements. The
total observations possible will vary due to analyzer response time, lag
times for time sharing systems, and duration of exposures.
3.2.2. QUALITY CONTROL CHECKS
3.2.2.1. ZERO AND SPAN CHECKS
Zero and span checks (OSC) will be used to assess data from automated
and manual methods for precision. Each analyzer used to measure SO2, 0^
and NO must have a weekly one-point zero and span check conducted with
the vaifues recorded on the zero and span data sheet.
Zero checks should be made by attaching the sample inlet line of the
analyzer to a zero air source, such as an activated charcoal filter and
silica gel, and allowing the analyzer to come to equilibrium and then
recording the analyzer's output. Span checks should be made by attaching
the sample inlet line of the analyzer to the appropriate outlet of a
calibration source and allowing the analyzer to equilibrate and then
recording the output. The concentrations of the calibration gas from the
source should be in the mid-range of the expected level of pollutant to be
measured. Using two check points within the experimental range instead of
just the midpoint is recommended. Care must be taken to insure the outlet
of the calibrator is vented to the atmosphere to avoid excessive back
pressure. Also, the outlet flow from the calibrator must be greater than
the inlet flow of the instrument to avoid dilution from the ambient air.
Changes or adjustments in either the zero or span settings after the
initial startup of analyzers must be made when the reading obtained is
greater than ± 5% of the known value of the zero or calibration gas. If
this occurs, values on data sheets should be noted and brought to the
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attention of the site project leader so that corrective action can be
initiated immediately.
3.2.2.2 MULTIPOINT CALIBRATION
With a change of greater than ± 10% in the zero or span check
(mentioned above), the operator should perform a multipoint calibration
(MPC) on the analyzer, using at least four points including zero. If the
slope of the new MPC curve differs significantly (approximately 10X) from
the previous slope of the MPC curve, then the analyzer will be removed
from service and repaired. However, it is first necessary to assure that
differences in the MPCs are not caused by inaccurate readings, errors in
transcribing, or in the calculations.
It is necessary to perform a multipoint calibration after an analyzer
has malfunctioned, since the required repair may have affected the
calibration. The MPC should be recorded in the analyzer's logbook along
with the cause of the malfunction and the repair procedures. An MPC will
be run at each startup of the analyzers. Calibrators and monitors should
have an external calibration check and audit at least once annually on
site.
3.2.2.3. CALIBRATION GAS SOURCES
Each site will have a calibration system for generating known levels
of the pollutant gases. These systems will be used in making multipoint
calibration curves and zero and span checks.
The gaseous standards, such as permeation tube devices and cylinders
of compressed gases, that are used to obtain the test concentrations for
SO2 and NO2 must be working standards traceable to a National Bureau of
Standards gaseous Standard Reference Material (SRM) and used only within
the certified period.
Test concentrations for 0, can be supplied by the use of a UV lamp
installed in a calibration system that has the capability of supplying 0^
to that analyzer in constant concentration at variable levels.
Test concentrations of the calibration gases must be supplied to the
analyzer so that the analyzers are operating in their normal sampling
mode. Test gases should pass through as much of the air line system as is
practicable.
Verification of gas concentrations in the calibration cylinders
should be accomplished every 12 months by sending the cylinders to the
Performance Evaluation Branch of EPA's Quality Assurance Division of the
Environmental Monitoring Systems Laboratory (at Research Triangle Park,
North Carolina 27711) for analysis. Arrangements can be made by calling
919/541-2723 or FTS 629-2723.
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3.2.2.4. AUDITS
Yearly external or independent internal audits will be used to assess
data from automated and manual methods for accuracy. Independence is
achieved by using standards and equipment different from those routinely
used during calibration. It is preferred that audits be made by a
different operator/analyst than the one conducting the routine analysis.
If, however, circumstances dictate the routine operator/analyst also be
the auditor, the individual conducting the audit must not be provided
beforehand with the value of the standard used in auditing. Results of
such audits will be examined carefully to detect any bias. All analyzers
that measure SO^, 0^, and NC^ are audited in the same manner. That is,
each analyzer is challenged with a known concentration of pollutant gas in
concentrations at five levels including zero air. The differences between
the known concentrations and the measured analyzer values are used to
assess the accuracy of the monitoring data.
At least one audit should be conducted at each site during the
growing season in which the data are being collected. The audit should be
done approximately midway into the growing season. The acceptable
accuracy level for analyzers in these audits will be ± 15 percent. This
level is equivalent to the accuracy level established by EPA's Quality
Assurance Division of the Environmental Monitoring Systems Laboratory at
Research Triangle Park, North Carolina 27711.
Audit materials will be those furnished by the National Bureau of
Standards (NBS SRM) or those that can be traced directly to SRMs.
Calculations for the precision and accuracy of the measuring process will
be done in accordance with the requirements as stated in the Federal
Register, Vol. 43, No. 152, dated Monday, August 7, 1978, pages 34908 and
34909.
3.2.2.5. SAMPLING AND DATA COLLECTION SAMPLING SYSTEM
The sampling lines in the system will be inspected biweekly for
foreign material and cleaned or replaced if necessary. Residence time of
the sample gas must be less than 60 seconds. Sample inlet lines to the
analyzers will consist of material that does not disturb the sample's
integrity. Inlet sample filters are highly recommended. Losses of air
pollutants in the system should be calculated bi-annually. Delivery rates
should be adjusted to compensate for system losses. Flow meters should be
checked twice annually. FEP (fluorinated ethylene propylene) is
preferable over TFE (tetrafluoroethylene) due to the tendency of the TFE
to absorb SO2 and then degas after SO2 is removed from the air stream.
3.2.2.6. EFFICIENCY TESTS
Constant concentrations of a series of test gas outputs from a
certified source are used in determining sample system efficiency. A
recently calibrated (same day) analyzer is used in conducting the sample
system efficiency test in the normal sampling mode. The chamber's sample
line is attached to the source and a known concentration of test gas is
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generated and introduced into the sample line. The analyzer's response to
this test gas is then compared to the known concentration. The percent
difference between the site's analyzer output and the known concentrations
of test gas will be calculated and reported to the project leader for each
sample line in the system.
Other methods for determining the sample system efficiency may be
acceptable provided the concentration of the test gas remains constant
(±5%) throughout the tests and the concentration is at least eight times
the minimum detection level of the analyzer. A discussion of these
methods must be part of the quality assurance documentation kept at the
site.
These tests should be done at least prior to the start of the
exposures and after the completion of the study, or every six months,
whichever is less.
3.2.3. DATA REPORTING
The project leader, prior to data collection, will write procedures
for data collection and reporting to insure the comparability of the data
with other sites. Data collection from those analyzers and instruments
that are connected to a data acquisition system will be in a format
comparable with the research project as specified by the project leader.
Data will be reported in engineering units on those systems which have the
capability of converting voltage to the desired units. The engineering
units for the various parameters are as follows:
Ozone, SO2, N0x ppm (altitude influences may
be significant)
Solar radiation ...ymol cm s
Temperature °C
Humidity % relative humidity
Wind speed m s ^
Wind direction degree azimuth.
3.2.3.1. DATA COLLECTION FLOW CHARTS
Data flow charts giving the data collection format used at the
different sites should be standardized by the Forest Response Program
Cooperatives.
3.2.3.2. DATA VALIDATION
The senior scientist at each site will be responsible for data
validation. Data sheets, maintenance records, strip chart recording, and
other pertinent data will be examined for:
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° high or low values (outliers);
° rapid excursions, such as caused by electronic interference;
0 repetitious values, such as caused by equipment malfunction;
" time continuity; and
° completeness, at least 75% of the data available from the sampling
system in use.
The following references for data validation are available from CERL
upon request: Quality Assurance and Data Validation for the St. Louis
Regional Air Pollutant Study written by R.B. Jurgens and R.C. Rhodes, and
Screening Procedures for Ambient Air Quality Data, EPA-450/2-78-037 (OAQPS
1.2-092)7 July 1978.
3.2.4. ANALYZER METHOD REQUIREMENTS
All analyzers used in the project for measuring pollutant levels in
the ambient atmosphere must be a Reference Method or have met the
equivalency requirements as specified by EPA in the Federal Register, AO
CFR 53. Any modifications made to the air pollutant analyzers that
disqualify them as reference or equivalent methods (e.g., changes made in
order to perform special purpose monitoring as required by the research
project) must be noted in the instrument logbook..
3.2.5. AMBIENT AIR ANALYZERS - OPERATION AND MAINTENANCE
1. Operation procedures will be those specified in the
manufacturer's operators manual.
2. Manufacturer's operators manuals will be available for each type
analyzer at each location.
3. Measurement principles and interferences caused by CO, HjS, and
heated silver scrubbers are discussed in section 3 of Document
No. QAD/M-79.12: Summary of Performance Test Results and
Comparative Data for Designated Equivalent Methods for SO^. This
document is available upon request from the Quality Assurance
Officer at CERL.
4. A preventive maintenance schedule will be followed by the
operators for each analyzer, instrument, and system used in the
collection of data for the project.
5. An example of the forms will be included in each analyzer's
operators manual. They indicate what needs to be done and the
frequency.
6. To insure implementation of the preventive maintenance schedule,
the forms will be filled out by the operator and a copy sent to
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the QA Officer, and the Project and/or Field Leader who will have
the responsibility of assuring the maintenance is being
performed. Originals are to be stored on site.
7. A supply of spare parts required for routine maintenance of each
analyzer, instrument, and system will be maintained on site to
insure that downtime is held to a minimum.
8. A logbook for each analyzer, instrument, and system will be
maintained and all malfunctions, repairs, multipoint
calibrations, modifications, etc. will be noted.
3.3. REFERENCES
Jurgens, R.B. and R.C. Rhodes. 1976. Quality Assurance and Data
Validation for the St. Louis Regional Air Pollutant Study. In:
Proceedings of the Conference on Environmental Modeling and
Simulation, V. R. Ott, ed. EPA 600/9-76-016.
U.S. Environmental Protection Agency. 1978. Screening Procedures for
Ambient Air Quality Data. EPA-450/2-78-037 (OAQPS 1.2-092).
Summary of Performance Test Results and Comparative Data for Designated
Equivalent Methods for SO2. 1979. QAD/M-79.12. (Complete reference
unavailable.)
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4. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OP NET CARBON EXCHANGE
4.1. SCOPE AND PURPOSE
Several previous studies have indicated that atmospheric pollution
impacts the physiological processes involved in net carbon exchange (NCE)
and may ultimately reduce forest productivity. Measurements of NCE can
help identify:
0 the extent to which photosynthesis and respiration are impacted by
dry and wet deposition and
0 can provide information to use in estimating the growth loss that
may result from pollutant damage to the physiological processes
involved in NCE and leaf area dynamics.
This SOP will outline the minimum SOPs for relating NCE of seedlings
grown in growth chambers or open-top chambers to (1) NCE capacity at
specified environmental conditions and (2) to relate measures of NCE and
leaf area to seedling growth rate.
The NCE measurements considered are those necessary to meet the
objectives outlined in the 1986 Forest Response Program.
4.2. MATERIALS AND SUPPLIES
4.2.1. EQUIPMENT
Any NCE equipment which meets the specifications given in Appendix B
may be used. Portable units commercially available include the LI-COR
LI-6000 and ADC-LCA2. If response curves for NCE to carbon dioxide
concentration, light, temperature and relative humidity are required,
cuvettes with the capacity for manipulating these elements will be
required. It will be the responsibility of the operator to be sure the
appropriate chamber is selected so that leaf temperature elevation, C0£
depletion, and boundary layer resistance are maintained within the
specified limits given in Appendix B.
4.2.2. CHEMICALS/REAGENTS
0 Primary standard gases 0, 350, and 1000 ppm that are traceable to
NBS standards
° Soda lime
° Magnesium perchlorate
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4.3. PROCEDURES
NCE measures are instantaneous readings which are not amenable to
precision and accuracy statements and for which no standards are
available. Quality assurance largely depends on calibration and spot
checks during equipment operation to ensure the equipment is functioning
properly. This section deals with what plant material should be measured,
the frequency of measurements needed for two specific type studies, and
what associated environmental variables should be measured so that data
can be compared across locations.
In general, the operator should follow the manufacturer's
standardization checks and procedures (Appendix B) to ensure proper
operation and maintenance of instruments used in making the NCE and
associated environmental measurements.
4.3.1. SAMPLE PREPARATION
0 Use random selection procedures to select trees and branches for
measurement of NCE as dictated by experimental design.
° Select foliage from each morphological class (flush, level of
maturity, age) as dictated by experimental design.
0 Mark foliage to be enclosed in the chamber so it can be reused for
the remainder of the day. This is required to minimize the amount
of leaf area determination that has to be made.
4.3.2. EQUIPMENT OPERATION
4.3.2.1. MEASUREMENT LOCATION ON SELECTED FOLIAGE
4.3.2.1.1. Long Needle Conifers-Pine Like
Center the cuvette on the selected needles as much, as possible if the
entire needle is not enclosed in the chamber.
4.3.2.1.2. Short Needle Conifers-Spruce Like
Center cuvette on branch containing foliage in the age class to be
measured.
4.3.2.1.3. Hardwoods
Center the cuvette on the side of leaf to be measured.
4.3.2.1.4. Alternative
Entire seedling may be enclosed in the chamber. In this case, and in
the case of short needle conifers, the amount of stem and branch material
enclosed in the cuvette should be determined.
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4.3.2.2. CHAMBER FOLIAGE CONSIDERATIONS - FOLIAGE QUANTITY AND PLACEMENT
IN THE CHAMBER
4.3.2.2.1. Portable Units
Long Needle Conifers-Pine Like
1. Use 2-5 needles (one fascicle) - this will depend on pine type.
2. Place needles so they do not run diagonally across the chamber if
needle length is to be assumed to be equal to the length of the
chamber.
3. Place needles in the cuvette so they do not overlap.
4. Place needles so they do not lay against each other.
5. Program instruments with a built-in memory vith an estimate of
the actual needle surface area to be enclosed in the chamber.
This will help the operator to know if the instrument is within
an expected range of operation.
Short Needle Conifers-Spruce Like
1. The length of the chamber and needle length and frequency will
dictate the amount of foliage that is included in the chamber.
2. Caution: Boundary layer resistance problems can be created when
large amounts of foliage are used (see Appendix B).
3. Program the equipment memory with a fixed value of leaf area and
adjust carbon exchange rates as soon as leaf area is determined.
Hardwoods
1. It is best to use a chamber which provides a fixed leaf area
inside the chamber.
2. Program the equipment memory with the actual leaf area enclosed
inside the cuvette.
4.3.2.2.2. Controlled Environment-Large Cuvette Units
0 The amount of foliage enclosed in the cuvette will depend on the
cuvette size and flow rates used.
0 Placement should be such that foliage overlap and self shading are
minimized and conditions specified in Appendix B are met.
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4.3.2.3. DETERMINATION OF LEAF AREA ENCLOSED IN THE CUVETTE
This section outlines the steps recommended for determining leaf area
using either destructive or non-destructive techniques. Whether a
destructive or non-destructive technique is used will depend on the
available plant material and whether the NCE measurement coincides with a
planned destructive harvest. The actual procedures for determining leaf
area are covered in the SOP for Measurement of Seedling Leaf Area (section
6.3). The technique selected for determining cuvette leaf area must meet
the specification outlined in Appendix B and follow the QA checks outlined
in the SOP for Measurement of Seedling Leaf Area.
4.3.2.3.1. Non-Destructive Method
Non-flat needle Conifers
1. Determine the number of needles in the cuvette for each sample.
2. Determine the length of all needles enclosed in the cuvette.
3. Determine the diameter on a subsample of needles.
4. Develop a length and/or width equation for predicting area of all
needles.
5. Estimate leaf area from an equation relating leaf area to needle
length and/or width measurements.
An alternative to steps 3, 4, and 5 is to relate a single dimension
measurement (e.g., length) to volume displacement and2then calculate total
surface area. To use either of these techniques, an R of .90 or better
should be obtained for the relationship between the measured dimension
variable(s) and measured leaf area.
4.3.2.3.2. Destructive Method
Non-flat needle Conifers
1. Harvest foliage enclosed within the cuvette.
2. Determine leaf area by a volume displacement method.
Hardwoods and Flat-needle Conifers
For hardwoods and flat-needle conifers, projected leaf area will be
determined by a planimetric technique such as with the LI-3000, LIr3100,
or Delta-T devices.
4.3.2.4. FREQUENCY OF MEASUREMENT DATES
The actual number of days between measurements of NCE cannot be
specified for all study objectives and conditions. However, some
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consideration should be given to the maximum number of days that should be
permitted to elapse before NCE measurements are repeated for two general
objectives that are of major importance to the Forest Response Program.
These two objectives are (1) determination of the impact of pollutant
exposure on the physiological processes involved in NCE and (2)
determination of the relationship between pollutant exposure NCE and
seedling growth rates.
4.3.2.4.1. For Studies with the General Objective of Relating Pollutant
Exposure to Impacts on the Physiological Processes Involved
in NCE
For pollutant exposure studies conducted in growth chambers and that
are less than four months in duration, NCE should be measured (1) prior to
the beginning of the experiment and (2) at the end of the experiment. Two
general conditions should be met for these measurements of NCE:
1. Prior to measurement, the seedlings should be given at least one
week to adjust to the environmental conditions under which NCE is
to be determined (e.g., NCE should not be determined at full
sunlight if the seedlings are kept in a facility where they
normally receive only one-half full sunlight).
2. Environmental conditions (C0„, light, temperature) should be the
same for both measurement dates if the investigator is interested
in determining the change in NCE capacity over the exposure
period.
For exposure periods longer than four months, NCE should be measured
every four months and at the end of the experiment. Measurement
considerations are the same as given for the short term studies. In the
case of open-top chamber studies, measurement conditions will not be the
same for each measurement date.
4.3.2.4.2. For Studies with the General Objective of Determining the
Effect of Pollutant Exposure on NCE and Relating Changes in
Whole Plant NCE to Seedling Growth Rates
Net carbon exchange must be measured at a minimum on a two week
basis.
4.3.2.5. FREQUENCY OF MEASUREMENTS WITHIN A MEASUREMENT DATE
4.3.2.5.1. For Studies with the General Objective of Relating Pollutant
Exposure to Impacts on the Physiological Processes Involved
in NCE
Measure NCE at one time period during the photoperiod. This should
be when light, temperature, and C0„ are closest to their optimum value
that is provided by the facility at which the study is being conducted.
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Measure NCE at one time during the night period after the seedlings
have been in the dark for at least one hour and temperature has been
stable for one hour.
4.3.2.5.2. For Studies with the General Objective of Determining the
Effect of Pollutant Exposure on NCE and Relating Changes~Tn
Whole Plant NCE to Seedling Growth Rates
NCE potential should be measured over the entire range of light,
temperature, and relative humidity conditions that can occur at each
measurement date. This can be accomplished two ways:
1. by constructing light and temperature response curves while
controlling relative humidity, or
2. by measuring NCE on both cloudy and clear days at 3 hour
intervals throughout the day.
Note: Both approaches take considerable amounts of time, and it is
unlikely that more than 1 or 2 families could be considered for
this type study. It will require that a minimum of 5 trees be
measured for each treatment-family combination.
4.3.2.6. REQUIRED ENVIRONMENTAL, PLANT, AND INSTRUMENT MEASURES TO BE
TAKEN WITH EACH SAMPLING PERIOD WITHIN A DAY
0 Ambient CO2 concentration
0 Absolute chamber CO2
0 Leaf temperature (This will not be possible with conifers -
record chamber air temperature.) Note: caution given in
Appendix B.
0 Air temperature
0 Chamber relative humidity
0 Air relative humidity
0 Photosynthetically active radiation
0 Stomatal conductance
° Flow rate
0 Barometric pressure
4.4. PREVENTIVE MAINTENANCE
The following guidelines apply to the cuvette and the infra-red gas
analyzer.
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0 Use and store per guidelines given in the operators manual
provided by each company.
6 Do not leave exposed to direct sunlight when not in use.
0 Do not store under environmental conditions that fall above or
belov the operating range of the instrument or that vary
widely.
0 Do not use when weather is misty or raining - carry a protective
covering with you to cover the instrument if it begins to rain.
0 Do not lay chamber on rough surfaces or it will get scratched
and light transmittance properties will decrease.
4.5. CALIBRATION PROCEDURES
4.5.1. C02 ANALYZER
0 Factory calibrate annually.
° Zero check and readjust hourly during measurement sessions.
0 If zero drift is >5 ppm in three consecutive hourly checks, return
the instrument to the site supervisor for repair.
° If a zero drift of >5 ppm occurs, requiring adjustment, recheck
and readjust the span check also before continuing to use the
instrument.
° Span check at the beginning of each measurement day using primary
standard gases with 0 (N„) and 350 ppm (CO^ in that are
traceable to NBS standards.
4.5.2. QUANTUM SENSOR (LI-190 S-l)
° Factory calibrate annually.
° Keep protective cover on the sensor when not in use.
4.5.3. FLOW METER
0 Factory calibrate annually.
° Check every four months with a high quality rotometer mounted in
series with the air stream entering the cuvette. If they disagree
by more than 6%, the site supervisor should be notified and
repairs made before the instrument is reused.
4.5.4. RELATIVE HUMIDITY SENSOR
0 Factory calibrate annually.
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0 Spot check monthly with air of tvo known vapors (high and low
humidity) within the operating range of the instrument. A dew
point hygrometer that has been calibrated within the measurement
year or spot checks kits available from LI-COP for the LI-1600 may
be used. If the measured values differ by more than 5% but less
than 10X from the standard values, readjust the instrument and
record the adjustment in the log book. If the deviation is
greater than 10%, then (1) replace the sensor and recalibrate or
(2) send to the factory for recalibration.
4.5.5. ¦ CUVETTE THERMOCOUPLE OR THERMISTORS
0 Factory calibrate annually.
° Compare readings at a high and low temperature with a high quality
thermometer that has been calibrated by the water bath technique
on a six month interval. If readings differ by more than ±2°C,
then readjust and recalibrate the instrument.
° For units with a thermistor housed within the unit and in the
cuvette chamber, a comparison of the thermocouple readings when
the chamber is open and the fan running is a good field check.
0 All calibrations performed should be recorded, dated, and signed
in an instrument log book. This does not include field spot
checks.
4.6. CALCULATIONS/UNITS
4.6.1. CALCULATIONS
° Calculations are to be done as recommended by the manufacturer's
manual. In the case where no manual is available, refer to pp.
50-54 and pp. 162-166 in Plant Photosynthetic Production - Manual
of Methods by Sestak, Catsky and Jarvis (1971) for NCE
calculations. For conductance calculation of CO^, 0^, and 0y
follow the procedures outlined by Coombs et al., (1985) using the
appropriate diffusivity coefficients (refer to pp. 85-87).
° Calculations should be done on a per leaf area and an oven dry
weight basis. Leaf area for conifers will be that of the total
surface area. Leaf area for hardwoods will be that for one
surface. If stomatal conductance of the adaxial surface is >.04
cm/s it should be reported in the written report for the study.
4.6.2. UNITS
Apparent net photosynthesis (NCE)
Dark respiration
-2 -1
.CO2 ymol m s
-2 -1
.COj umol m s
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Leaf Area 2
Conifers.... cm (total surface)
2
Hardwoods cm (one sided)
COj concentration ppm
Temperature ,.. . . °C
-2 -1
Photosynthetically active radiation ymol m s
-2 -1
Transpiration.... ymol m s
-2 -1
Stomatal conductance ymol m s
Relative humidity %
Leaf area cm^
Barometric Pressure KPa
4.6.3. RECORDING FORMAT (see Appendix D for suggested computer codes and
reporting symbols).
Date 0-365
Time Standard Military
Treatment Number
Tree Number
Branch-Sample Number
Foliage age class 01dest=l, Next Age=2,
Etc.
2
Sample leaf area Nearest hundredth cm
2
Programmed value Nearest hundredth cm
2
Actual value Nearest hundredth cm
Flow rate 1/hr
Ambient C02 Nearest ppm
Chamber CO2 Nearest ppm
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Leaf temperature Nearest tenth °C
Air temperature Nearest tenth °C
Chamber relative humidity Nearest percent
Air relative humidity Nearest percent
-2 -1
Photosynthetically Active Radiation.. Nearest umol m s
-2 -1
Carbon exchange rate (NCE) Nearest pmol m s
Internal COj concentration Nearest ppm
Transpiration rate Nearest umol
Stomatal conductance Nearest ymol
4.7. ERROR ALLOWANCE AND DATA QUALITY
A.7.1. EQUIPMENT PRECISION AND ACCURACY REQUIREMENTS
Instrument/Procedure Accuracy Precision
Quantum sensor
±5%
±2%
Humidity sensor
±5%
±2%
Temperature sensor
±1°C
±1°C
CO2 analyzer
±5 ppm
±2 ppm
Noise
< 1%
N/A
Area meter
±5%
±5%
Scales/oven dry weight
±1.5%
±1.5*
All instrumentation will be checked to ensure that precision and
accuracy are within the acceptable range. Precision measures for the CC^
analyzer and associated measures are to be made at the time of calibration
under controlled conditions.
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4.7.2. DATA QUALITY OBJECTIVES
Repeated Measurement Measurement
Reporting Error at Accuracy
Variable Units Lover Limit Upper Limit Tolerance
Photosynthesis
pmol
-2 -1
m s
+
10*
+
10*
15*
Transpiration
Mmol
-2 -1
m s
+
10*
+
10*
15*
Needle Conductance
umol
-2 -1
m s
+
10*
+
10*
10*
Respiration
Vimol
-2 -1
m s
+
10*
+
10*
15*
A.8. REFERENCES
Coombs, J.D., D.O. Hall, S.P. Long, and J.M.O. Scurlock (eds.). 1985.
Techniques in Bioproductivity and Photosynthesis. Pergamon Press, New
York. 298 pp.
Sestak, Z.,J.Catsky and P.G. Jarvis. 1971. Plant Photosynthetic
Production - Manual of Methods. W. Junk Publications, The Hague.
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5. STANDARD OPERATING PROCEDURE FOR PLANT WATER RELATIONS MEASUREMENTS
5.1. SCOPE AND PURPOSE
Plant water relations measurements are important measures to include
in the Forest Response Program (FRP) because they (1) serve as a good
index of plant vigor and (2) are a necessary component in estimating the
amount of atmospheric pollutant that was taken in by the plant under
study. The two plant water relations measures to be addressed in this SOP
are (1) measures of xylem pressure potential (XP) and (2) measures of
stomatal conductance (i.e., leaf water vapor exchange). The procedures
described will be for (pine) seedlings used in growth chamber or open-top
chamber studies as outlined for the Forest Response Program.
5.2. MATERIALS AND SUPPLIES
5.2.1. EQUIPMENT
5.2.1.1. XYLEM PRESSURE POTENTIAL DETERMINATION
0 Pressure Chamber - The pressure chamber is the recommended
equipment for determining xylem pressure potential. Two available
sources are PMS Instruments and Soil Moisture Test.
0 Dry nitrogen source and dual stage pressure regulators
0 Tank wrenches
° Rate valve adjustment wrench
0 High intensity light source for pre-dawn measurements; also extra
batteries
° A 10X or better magnifying glass; alternatively, a binocular scope
mounted on the pressure chamber
° Industrial grade razor blades
° Zip lock plastic bags
° Paper towels
° Plastic wrap
° Tissues
° Water
° Stem inserter
° External light source and extra batteries and bulbs
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5.2.1.2. LEAF WATER VAPOR EXCHANGE DETERMINATION
° Porometer - The recommended method of measurement of stomatal
conductance is by the steady state porometer technique.
Instruments such as the LI-1600, Delta-T MK.3 porometer, LI-6000,
and LCA-2 have the capability of making these measurements.
° Cuvette Chambers - The cuvette chamber should meet the
specifications outlined in Appendix B. For pine-lilce foliage, a
small volume square chamber has several advantages: (1) readings
can be taken on a single fascicle - this makes foliage placement
into the cuvette easy and fast; (2) the length of the chamber can
be taken as the length of the needle; (3) the response time of a
small volume chamber vill be fast, thus minimizing temperature,
relative humidity, and C0^ departure from ambient; (A) only small
amounts of foliage are required, thus keeping the amount of
foliage for which leaf area has to be determined to a minimum.
° Desiccant such as silica gel
° Extra batteries
5.2.2. CHEMICALS/REAGENTS
5.2.2.1. XYLEM PRESSURE POTENTIAL DETERMINATION
0 Alcohol for cleaning resin on equipment
° Vaseline for greasing 0-rings
5.2.2.2. LEAF WATER VAPOR EXCHANGE DETERMINATION
0 Silica gel
5.3. PROCEDURES
5.3.1. SAMPLE PREPARATION
5.3.1.1. XYLEM PRESSURE POTENTIAL DETERMINATION
5.3.1.1.1. Sample Marking
1. Set pressure chamber up as close to seedling to be measured as
possible.
2. For predawn determinations, select and mark on the day prior to
the measurement day the foliage sample is to be used. The marking
should contain the key sample identification information. When
sample is collected put ID tag vith the sample.
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5.3.1.1.2. Sample Collection
1. If vapor pressure deficits are greater than 10 mbar (.001 MPa),
wrap the foliage to be removed with plastic wrap just prior to
cutting it off. This will prevent rapid changes in XP from
occurring before determinations can be made.
2. Remove the petiole, fascicle, or branch from the seedling.
3. If petioles or branches are used for XP determination, they should
be cut at a 45° angle with a sharp razor blade free of resin. The
45° angle creates a surface that is easier to illuminate and
detect the end point. A sharp clean razor blade will keep the
petiole or stem from splitting during the excision process. If
stems are to be used, cut the stem so that a distance of 3 cm is
left between the cut surface and the first lateral branch.
4. If fascicles are to be used, they should be cut as close to the
stem source as possible. On some species it may be better to tear
the fascicle off; this leaves the vascular trace intact.
Note: Accuracy and precision considerations — Accuracy and
precision will be greater for stem material than for fascicle
than for individual needles. This is especially true for the
predawn determinations of XP. The available study material
will dictate what material can be used. Determination of XP on
individual needles is not recommended due to high sample to
sample variation.
5.3.1.1.3. Storage of Samples
1. In some studies it may be possible to cover the seedlings with a
bucket to extend the time period available for predawn XP
determination.
2. In other studies it will be necessary to collect multiple samples
and transport the material for XP measurements. If sample has to
be transported or held for more than 30 seconds after cutting, it
should be placed in a zip lock bag (or similar device) which
contains a moist paper towel (no excess water). All excess air
should be squeezed out of the bag and then the bag sealed. Store
the bag in a cool spot out of direct light. Storage time should
be less than 5 minutes. If longer storage times are required, a
study which quantifies the magnitude of change in XP that occurs
in storage over the length of storage period required should be
made and measured XP adjusted for length of storage time.
5.3.1.2. LEAF WATER VAPOR EXCHANGE DETERMINATION
1. Mark foliage to be used for water vapor exchange. This will be
necessary so foliage leaf area can be determined and to locate the
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sample of repetitive measures within the day or to be made on the
same foliage.
2. Sample selection procedures should, be the same as those outlined
in the SOP for Net Carbon Exchange (section 4.3).
3. If samples are to be collected and stored for leaf area
determination, they should be placed in a zip lock bag and placed
in the refrigerator. A tag with all key identification
information must be placed inside or on each bag. Samples should
not be stored more than 2-3 days or decomposition may be
significant.
5.3.2. EQUIPMENT OPERATION
5.3.2.1. XYLEM PRESSURE POTENTIAL DETERMINATION — PRESSURE CHAMBER
1. Collect foliage as per Section 5.3.1.
2. On stem material, peel the bark back about 2.54 cm to prevent
"foaming"-material from the phloem-cambium area from bubbling
onto the cut surface. Do not tear any small lateral branches off
the sample in this step.
3. Insert sample material into the chamber stopper or holder. Use
an inserter where necessary. If the stem or fascicle is
inadvertently bent sharply do not use it. Note, under vapor
pressure deficits greater than 10 mbars (.001 MPa), keep the
material wrapped until the insertion step is complete.
4. For stems, insert the peeled stem through the stopper and lid so
that = 6 mm of stem is above the upper lid surface.
5. Insert sample and pressurize the chamber.
6. Begin with chamber valve off and rate valve off.
7. Put stopper vith sample inserted through it into the chamber lid.
8. Remove plastic wrap from the foliage and fasten lid securely on
the chamber as quickly as possible.
9. Switch the chamber valve to the fill position.
10. Adjust rate valve to pressurize the chamber at .05 MPa (.5
bars)/second. For predawn XP measurements when soil moisture is
high, a rate about .025 MPa/s may be desirable. When it has
already been established by previous measurements that XP is low,
a faster pressurization is permitted until a reading within > .5
MPa is obtained. Then the rate should be slowed to 0.05 MPa/s.
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11. For species with resin ducts such as the pines, during the
pressurization process it will be necessary to periodically wipe
the resin off the cut surface with a tissue that has a high
absortivity capacity.
12. View end point through a 10X or greater magnifying glass or
scope. The end point is when the xylem surface first wets.
13. Turn chamber valve to off.
14. Turn rate valve off.
15. Turn chamber valve to exhaust.
16. Let gauge pressure go to zero.
17. Remove lid. Turn chamber valve to off.
18. Dispose of sample.
Note: Accuracy and precision considerations — For plant water
relations, the thermocouple psychrometer is the accepted
standard of measure for plant water potential. It would be
possible to compare xylem pressure potential measurements to
plant water potentials as measured with the thermocouple
psychrometer. However, since the objective of plant water
relations measures outlined for the FRP is to obtain an index
of plant moisture states, this is not justified. Thus no
measure of accuracy will be made. A measure of precision can
be made by taking repeated measures of xylem pressure
potential using tissue from a single branch and using plant
material as similar as possible. This measure of precision
will still have a within-plant variation component in it as
well as a machine repeatability component. Precision of XP
determinations should be made for each species across the full
range of xylem pressure potentials that are experienced during
the study.
5.3.2.2. LEAF WATER VAPOR EXCHANGE — POROMETER/CUVETTE
For foliage selection, measurement location, and determination of
cuvette leaf area, see Net Carbon Exchange SOP (Section 4.3.1.-4.3.2.3.)
5.3.2.2.1. Frequency of Measurement Dates
This will be dictated by the rate of change of factors which are
known to influence stomatal functioning: (1) foliage development, (2)
vapor pressure deficits, (3) light levels, and (4) soil moisture supply.
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5.3.2.2.2. Porometer Operating Procedures: Checkout
1. Prior to each day's use of the porometer, freshly dried desiccant
is installed.
2. The system should then be checked for leaks by setting the flow to
zero.
3. Valves are then checked to see if they will fully open by turning
the flow meter to the fully open position.
5.3.2.2.3. Porometer Operating Procedures - Measurements
1. Operate per operators manual instructions (Appendix B).
2. Vith hardwoods, make sure the thermistor or thermocouple is
touching the lower surface of the leaf.
3. With conifers, make sure the thermistor or thermocouple is not
exposed to direct sunlight.
A. Associated light readings should be taken with the Quantum sensor
(held level).
5. Stomatal conductance readings should be made at a chamber relative
humidity that is within +5% of that observed for the ambient air.
6. Under high temperature and radiation conditions it may be
necessary to shield the chamber to keep chamber temperature during
the reading within +2°C of ambient air temperature.
7. For open-top studies, if dew is present it will not be possible to
measure stomatal conductance until the foliage has completely air
dried.
Note: Accuracy and precision considerations — It is not possible to
measure the same foliage and get an estimate of the precision
of the porometer measures because stomatal response to
repeated measurements does occur. Both the precision and
accuracy determinations will have to be made during, and will
depend on, proper calibration, operating procedures, and
equipment maintenance.
5.3.3. REQUIRED PLANT AND ENVIRONMENT MEASURES FOR ALL PLANT WATER
RELATIONS STUDIES
0 Air temperature
0 Relative humidity
0 Leaf temperature (for hardwoods)
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° Light intensity - net radiation or photosynthetically active
radiation.
0 Soil water potential - optional but desirable
Use air temperature and relative humidity to calculate vapor pressure
deficit for conifers. Use leaf temperature and relative humidity to
calculate vapor pressure gradient for hardwoods. For chamber studies or
measurements made below the canopy, use the Quantum sensor.
5.4. PREVENTIVE MAINTENANCE
5.4.1. PRESSURE CHAMBER
° Clean foliage and grit from around the chamber lid.
° Keep all hose and quick-disconnect fittings free of grit.
0 Keep the 0-rings free of resin and greased with a lubricant.
° Do not run the rate valve down hard against the valve seat.
0 Do not leave foliage in the chamber during storage. Make sure the
chamber is cleaned and lubricated before storage.
0 Store the magnifying glass where it will not be scratched. Clean
all resin from the lens before storing.
° Remove batteries from flashlights before storing.
0 Dispose of all used razor blades at the end of the measurement
day.
° Store pressure regulator in a clean environment.
5.4.2. P0R0METER
Storage procedures are the same as those given for the LI-6000 in the
SOP for Net Carbon Exchange (Section 4.4) and provided by the manufacturer
(Appendix C).
5.5. CALIBRATION PROCEDURES
5.5.1. PRESSURE CHAMBER
° No annual calibration is required.
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° Pressure gauge should be observed for jerky movements during the
pressurization process. If this occurs, have it replaced. The
pressure gauge should be checked to see that it returns to zero
when the pressure is released. If it does not, adjust the gauge
to properly read zero.
5.5.2. P0R0METER
0 An annual company calibration is required. The tests performed in
this calibration is given in Appendix E.
° Spot checks of the humidity sensor, Quantum sensor, and thermistor
are the same as those given in the Net Carbon Exchange SOP
sections 4.2.-4.5.and Appendix B.
5.6. CALCULATIONS/UNITS
Calculations of stomatal resistance, conductance, and transpiration
should be made as described in the porometer owner's manual.
5.6.1. UNITS
-2 -1
Stomatal conductance pmol m s
-2 -1
Stomatal resistance ymol m s
Transpiration vimol m ^s ^
-2 -1
Quantum flux density Vimol m s
Air temperature °C
Leaf temperature °C
Relative humidity %
Vapor pressure deficit or gradient mg/m
Soil water potential MPa
5.6.2. RECORDING FORMAT (See Appendix D for suggested computer codes and
reporting symbols.)
Date
0-365
Time
Standard Military
Treatment
Number
Seedling
Number
Sample
Number
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Xylem pressure potential MPa-nearest hundredth
Stomatal conductance nearest ymol
Stomatal resistance nearest jjmol
Transpiration nearest ymol
Quantum flux density nearest ymole
Air temperature °C-nearest tenth
Leaf temperature °C-nearest tenth
Relative humidity %-nearest tenth
_3
Vapor pressure deficit or gradient mg m -nearest tenth
Soil water potential MPa-nearest hundredth
Cuvette leaf area
2
Hardwoods nearest tenth cm (one
sided measure)
2
Conifers nearest tenth cm
(total surface)
5.7. ERROR ALLOWANCE AND DATA QUALITY
5.7.1. XYLEM PRESSURE POTENTIAL DETERMINATION
The major source of error in determining xylen pressure potential is
in reading the "end point." This error can be minimized by (1) following
the steps outlined in the operating procedure section (5.3.2.1.) of this
SOP and (2) providing appropriate training of new users before they are
allowed to make study measurements. The accepted precision for xylem
pressure potential determinations is +0.1 MPa.
5.7.2. LEAF WATER VAPOR EXCHANGE DETERMINATION
The error sources of steady state porometers arise from measurements
of temperature, relative humidity, flow rate, and leaf area. The
acceptable limits are the same as those given in Appendix B.
5.7.3. WATER RELATIONS MEASUREMENTS - DATA HANDLING
The QA procedures outlined for NCLAN biological measurements are
acceptable for the Forest Response Program (Appendix F).
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5.7.4. DATA QUALITY OBJECTIVES
Variable
Repeated Measurement Measurement
Reporting Error at Accuracy
Units Lover Limit Upper Limit Tolerance
Leaf Water
Potential
Leaf Water Content
Leaf Area
Needle Conductance
MPa
% wt
0.01 cm2
n -2 -1
pmol m s
+ 0.1%
+ 1.5%
+ 2 %
+ 10%
+ 0.1%
+ 1.5%
+ 5%
+ 10%
5%
1%
5%
10%
5.8. REFERENCES
N/A
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6. STANDARD OPERATING PROCEDURE FOR MEASUREMENT OF SEEDLING LEAF AREA
6.1. SCOPE AND PURPOSE
Measures of leaf area are necessary for calculating net carbon
exchange and water vapor exchange on a per unit leaf area basis.
Estimation of whole plant gas exchange and pollutant uptake requires
combining unit area rates of gas exchange with measures or estimates of
seedling leaf area. This SOP will address the procedure necessary for
determining leaf area by the planimetric and volume displacement methods.
6.2. MATERIALS AND SUPPLIES
6.2.1. EQUIPMENT
° vernier caliper
0 analytical balance (e.g., Mettler A30)
0 thin wire
0 leaf area meter
To determine leaf area using planimetric methods, several leaf area
meters can be used to give quick, reliable results with high resolution.
LI-COR, Inc., produces £he LI-3100 £rea Meter. It is an instrument with
interchangeable 1.00 mm and 0.1 mm resolution capability with a 35
and 105 mm lens. A 25 cm wide sample guide is provided for the 1.0 mm
resolution configuration. A 7.5 cm wide sample guide is available for the
0.1 mm resolution capability (LI-COR, 1979). Portable leaf area meters
such as the LI-3000 are available from LI-COR, Inc., for non-destructive
sampling and field work.
Another leaf area meter such as the LI-3000 used for studies in the
Forest Response Program is the Delta-T, distributed by Decagon Devices,
Inc. This system (Delta-T Devices, Ltd.) can measure the area of all
shapes and sizes of leaves, from large maple leaves and long cereal leaves
to small pine needles. Measurements of diseased and variegated leaf area
are possible if the discolored part is in good contrast with the remainder
of the leaf. Resolution^of l/300th of the scanned width and height is ^
given. Ranges from 1 mm for large areas (360 x 260 mm), down to 0.01 mm
, for small areas (35 x 26 mm).
6.3. PROCEDURES
6.3.1. SAMPLE PREPARATION
6.3.1.1. GENERAL CONSIDERATIONS FOR SAMPLE COLLECTION
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The amount and method of collection of material for leaf area
determination will depend on the objective of the experiment. However,
some general guidelines are worth noting.
° Foliage for which leaf area is to be determined should be
collected and placed in a sealed container and stored at
temperatures between 1.5-4.5°C if storage is required.
° Foliage should not be stored more than 4 days before leaf area
determinations are made or leaves will begin to deteriorate.
° The containers in which the foliage samples are stored should be
clearly marked with all pertinent treatment identification
information.
6.3.1.2. SAMPLE COLLECTION AND STEPS FOR DESTRUCTIVELY DETERMINING WHOLE
SEEDLING LEAF AREA
1. Identify the number of different morphological classes of needles
to be sampled (e.g., primary vs. secondary, first vs. second
flush).
2. Collect all foliage and identify by morphology class.
3. Select a 10% (fresh weight basis) subsample at random from each
morphology class.
4. Determine the leaf area of the subsample by procedures outlined
in Section 6.3.2.
5. Determine the oven dry weight (70°C) of the subsamples.
6. Determine oven dry weight (70°C) of all samples.
7. Use subsample measures of leaf ai^ea and oven dry weight to
determine specific leaf area (cm /g).
8. Multiply total dry weight of each morphological class by the
specific leaf area determined for each class to determine the
total leaf area of each seedling.
6.3.2. MEASUREMENT
6.3.2.1. MATHEMATICAL METHOD (DISPLACEMENT METHOD)
A procedure for estimating leaf area by using a mathematical
relationship between a leaf characteristic and total surface area has been
developed by Johnson (1984). As he describes in his publication on this
technique, "success lies in the ability to mathematically describe needle
geometry and to reproducibly measure needle volume to 0.01 ml using
Archimedes principle." Johnson assumes that
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"A pine needle in cross-section approximates a sector with
a radius, r, and an angle, 0. The angle, 0, is a function of
the number of needles per fascicle (0 = 360/n).
The volume of a sector is described by the following
equation:
V =
M
(1)
where V is volume (cm ), r is the radius of the sector (cm), n
is the number of needles per fascicle, and I is the length of
the needle (cm). The surface area of a sector is described by
A =
2r
2nr
(2)
To reduce the number of measurements, equations (1) and (2)
are first solved for r:
r =
Vn
JU
r =
21
2nZ
(3)
(A)
and then combined, solving for A, equation (5):
This is the final form of the total surface area equation
where A is the total surface ar^a (cm ), V is the displaced
volume of the needle sample (cm ), n is the number of needles
per fascicle, and £ is the comulative needle length of the
needle in the sample (cm)."
To determine the volume and radius, the following procedures were
used.
"As the fascicles were removed from a branch, they were
cut at the basal sheath and the entire needle sample of 20 to
30 needles was wrapped with a thin wire; The sample was
loosely wrapped with wire to avoid the trapping of air bubbles
around the needles, and if air bubbles were still a problem, a
surfactant can be added to the water. The volume of the
sample was then determined to the nearest 0.01 g by volume
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displacement on a Mettler A30 balance. The displaced volume
of the wire was measured and subtracted from the total volume.
Once the volume was measured, each needle in a sample was
individually measured for its length to the nearest millimeter
and for radius to the nearest 0.005 cm with a caliper.
...Two problems were encountered in attempting to measure
a representative mean radius: (1) needle taper varied with
the number of needles per fascicle, and (2) measurement error
was considerable due to needle compression by the caliper.
Needle taper was determined by measuring the radius at 2 mm
intervals along the length of a number of needles from each
species.... Using a caliper to measure needle radius proved
to be less than satisfactory due to the resolution of the
caliper (0.005 cm) and associated measurement errors resulting
from needle compression in the caliper. An error of 0.001 cm
in radius was calculated to result in a 3 percent change in
surface area.
— To measure a large number of needles, the fastest
method was to measure individual needle lengths in intact
fascicles. Length was measured from the top of the basal
sheath to the tip (rounding down to the nearest millimeter)
and then the needles were cut at the basal sheath prior to
measuring volume. These individual needle lengths were summed
to give a cumulative needle length for each sample." (Johnson,
1984).
6.3.2.2. PLANIMETRIC METHODS
Procedures for operating the LI-3100 follow:
"Connect the supplied power cord to the power input
connector at the rear of the instrument. A grounded three
prong wall connector is required for electrical service. Move
the 'on-off' switch to 'on.1 Press the "lamp start" firmly
and release after holding for approximately two seconds. If
the fluorescent tube does not illuminate, repeat the
procedure. Press the reset button to clear the display....
The display will rapidly accumulate numbers when the 'on-off'
switch is initially placed in the 'on' position. This
accumulation will continue until the fluorescent tube is
activated. If numbers continue to accumulate, then the
calibration screw should be adjusted counterclockwise until
the displayed numbers remain constant. Accurate or larger
than actual calibration disk measurements are normal at this
calibration screw adjustment. Data accumulation may continue
to occur if the fluorescent tube output is not sufficient (as
with initial starting in extremely cold conditions).
Excessively low line voltage is also a cause for continued
rapid spurious counting." (LI-C0R, 1979)
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In operating the Delta-T leaf area meter,
"... a TV camera views the object to be measured, which is
illuminated to contrast with its background. The Area Meter
Box sums the periods during which the line by line camera scan
is traversing an object. This sum is a measure of the area
and can easily be adjusted for calibration using an object of
known area. The output from the TV camera is displayed on a
monitor, together with a superimposed image of the actual
measured area. A digital display on the monitor screen shows
the area of the object in view, the number of measurements
made, and the total area.
An object will be detected and measured if the Area Meter
Box can identify it as being light or dark in relation to the
background. The Threshold Control sets the grey-level at
which an object will be detected. The effect of adjusting the
Threshold is seen on screen as a progressive blacking-in of
the object. When the whole shape is black the measured area
can be read off from the display. Once set for a particular
type of object the level does not need readjustment." (Delta-T
Devices, Ltd.)
Both leaf area meters are appropriate for broadleaf foliage where
surface area is primarily a function of leaf length and breadth.
Suitability for use on conifer foliage lessens because needle breadth
approaches needle thickness (Drew and Running, 1975). A regression or
conversion factor must be employed to relate projected surface area to
total surface area (Carlson and Johnstone, 1979) unless the needles are
flat.
6.3.2.3. GENERAL STEPS TO FOLLOW IN DETERMINING LEAF AREA BY THE
PLANIMETRIC METHOD
1. Let machine warm up 3-5 minutes.
2. Check the calibration (Section 6.5) before beginning a measure-
ment session. Use a calibration plate that is in the mid-range
of the area measurements to be made and with a similar
configuration as the foliage being measured. Take five measures
so both accuracy and precision can be determined.
3. For multi-needle conifer - clip the needles off just above the
fascicle bundle sheath.
4. Place the sample as perpendicular to the sensing element as
possible. This is only important for narrow leaf conifers.
5. Make sure foliage does not overlap.
6. After every 50 sample measurements, recheck the accuracy and
precision with a plate as described above for known area. If the
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reading of the calibration plate exceeds +5%, the belt should be
cleaned and adjusted if needed. If this does not reduce the
error to less than 5%, corrective action must be taken before
measurements proceed. A record of calibration checks and average
deviation of the readings from the known area plots must be
maintained.
6.4. PREVENTIVE MAINTENANCE
Maintenance for the LI-3100 involves cleaning of the belts, mirror,
and occasionally the camera lens. Clean the belts with water and a cloth
or absorbent paper. A detergent may be used for persistent contamination
but do not allow detergent to fall on the mirrors. Any scrubbing of the
mirrors to remove detergent spots may damage the mirror surface. Access
to the lover belt is facilitated by momentarily activating the "on-off"
switch to present surface near the sample tray. The inner surfaces are
cleaned by reaching into the access ports in the front plate. Loosen the
belts to facilitate access to the pulley surfaces.
If the mirror or camera lens must be cleaned, use the "blow brush"
provided with the instrument. If persistent dirt remains on the mirror,
use water and a soft absorbent paper such as lens paper.
Cleaning within the camera is not a frequent requirement but when
necessary, follow these steps. Remove the lens. Loosen the screws on the
outer camera pressure plate and lift the camera from the rails to more
easily inspect the interior. The camera remains connected so do not apply
tension to the connection. The rectangular sensitive device (RETICON) is
visible at the interior rear of the camera housing. Any speck o£ dirt on
this sensor will cause spurious counting. Use the "blow brush" provided
to remove dust. Do not place a moist cloth within the camera. The
adhesive dust retaining surface surrounding the RETICON and printed
circuit board would be damaged.
Maintenance for the Delta-T is similar.
6.5. CALIBRATION PROCEDURES
A warming period of 1-3 minutes may produce a calibration change of
approximately 1% from that obtained at the initial starting. This is a
fluorescent tube output response. Calibration should be performed 3-5
minutes after tube illumination.
Calibration is performed on both leaf area meters by placing a
calibration disk between the sample guides on the sample tray. Reset the
display and slide the disk onto the lower transparent belt and allow the
disk to travel through the instrument. Turn the "CAL" screw clockwise to
increase the displayed sample area. If the displayed area is too large,
adjust the "CAL" screw counterclockwise. When proper calibration has been
achieved, subsequent measurements of the calibration disk should result in
an error less than 5% of the actual area.
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6.6. CALCULATION/UNITS
2
The surface area of foliage is to be given in cm . For conifers, the
surface area is to be total surface area. This means all measures of
projected leaf area must be converted to total leaf area. For hardwoods,
the area of the foliage will be expressed as a single sided area.
6.7. ERROR ALLOWANCE AND DATA QUALITY
With optical planimeters, a dimensional correction factor must be
developed for each species measured. The precision of the LI-3100 was
determined at the 97% level with irregular-shaped complex objects. Most
applications will result in less error. A table of accuracy is shown
below (LI-COR, 1984).
2 2 2 2 2
Resolution 10 cm 5 cm 1 cm 0.5 cm 0.25 cm
1 mm2 „ + 1% +2% +5% + IX
0.1 mm + 0.5% + 1% +1% + 1.5% + 4%
For the Delta-T, resolution of l/300th of the scanned width and
height is achieved. It ranges from 1 mm for large areas (360 x
260 mms), to 0.02 mm for small areas (35 x 26 mms). Accuracy
ranges from 96-99%, depending on factors such as size, shape, and
contrast. The operator can maximize accuracy and resolution by
setting the camera to the lowest height permitted by the sample
dimensions, (so that the whole of the object remains within the
scanned area). Other significant factors affecting accuracy are
the amount of contrast between object and background, and whether
the object has features close to the resolution limit.
6.7.1. DATA QUALITY OBJECTIVE
Repeated Measurement Measurement
Reporting Error at Accuracy
Variable Units Lower Limit Upper Limit Tolerance
Leaf Area 0.01 cm^ +2% +5% 5%
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6.8. REFERENCES
Carlson, L.W., and V.D. Johnstone. 1979. Use of the rhizometer to
estimate foliar surface area. Can. For. Serv. Bi-monthly Res. Note
38:9.
Delta-T Devives, Ltd. Delta-T Devices: Scientific Instruments for
Ecology. 16pp. (Sole U.S. Distributor is Decagon Devices, Inc., NV
800 Fisk, Pullman VA 99163.)
Drew, A.P. and S.W. Running. 1975. A comparison of two techniques for
measuring surface area of conifer needles. For. Sci. 21: 231-232.
Johnson, J.D. 1984. A rapid technique for estimating total surface area
of pine needles. For. Sci. 30:913-921.
Kramer, P.J. and T.T. Kozolvski. 1979. Physiology of Woody Plants.
Academic Press. Nev York. 811 pp.
Kvet, J. and J. Marshall. 1971. Assessment of leaf area and other
assimilating plant surfaces, p. 517-555. In: Plant Photosyrithetic
Production - Manual of Methods, 2. Sestak, J. Catsky, and P.G. Jarvis
(eds.). V. Junk Publications, The Hague.
LI-COR. 1979. LI-3100 Area Meter Instruction Manual. LI-COR, Inc.,
Lincoln, Nebraska. 28pp.
LI-COR. 1984. Area Meters: LI-COR Instrumentation for Biological and
Environmental Sciences. LI-COR, Inc., Lincoln, Nebraska. 7pp,
Shelton, M.G. and G.L. Switzer. 1984. Variation in the surface area
relationships of loblolly pine fascicles. For. Sci. 30:355-363.
Thompson, F.B. and L. Leyton. 1971. Method for measuring the leaf
surface area of complex shoots. Nature 229:572.
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7. STANDARD OPERATING PROCEDURE FOR SEEDLING GROWTH MEASUREMENTS
7.1. SCOPE AND PURPOSE
Measures of seedling growth will be the major response variables used
to evaluate the impact of the assigned treatments to be tested under the
Forest Response Program. In many cases not only is the impact on total
productivity important, but also shifts in carbon allocation or growth
form that may result from these treatments are important. This SOP
outlines the procedures necessary to determine the weight change of the
foliage, stem, and root components for two type studies: (1) growth
chamber studies and (2) open-top chamber studies.
7.2. MATERIALS AND SUPPLIES
° Meter stick - graduated in cm
° Calipers
0 Analytical balance
7.3. PROCEDURES
7.3.1. GROWTH CHAMBER STUDIES
7.3.1.1. INITIAL SEEDLING MEASURES
1. Select 20 seedlings/family at random from the population that is
available for use in the exposure study.
2. Determine height from the root collar (original ground line) to
the tip of the bud with a meter stick.
3. Determine root collar diameter with a caliper at 1 cm above the
original root collar.
A. Separate each seedling into foliage, stem, and root components.
5. Determine oven dry weight (70°C) of each component.
6. Develop equations relating seedling height and diameter to
component dry weights.
7.3.1.2. ESTIMATING INITIAL WEIGHTS OF STUDY SEEDLINGS USING
RELATIONSHIPS DERIVED FROM SUBSAMPLE
1. Measure height and root collar diameter (as in 7.3.1.1) for all
seedlings going into the growth chamber.
2. Use the equations derived in 7.3.1.1 to estimate initial seedling
weight by components (foliage, stem, and root).
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3. Mark each container so subsequent measures of diameter and height
are made from the same side.
4. Note: Plant seedlings so that root collar diameter can be
measured at the same location as in 7.3.1.1.
7.3.1.3. ESTIMATING INTERIM AND FINAL WEIGHTS
1. Measure heights and diameters on a monthly basis using a meter
stick and calipers (optional).
2. At the final measurement date repeat all the initial steps
outlined in 7.3.1.1.
7.3.2. OPEN-TOP CHAMBER STUDIES
7.3.2.1. INITIAL SEEDLING MEASURES
1. Select 20 seedlings/family at random from the population that is
available for use in the open top chamber study.
2. Determine height from the root collar (original ground line) to
the tip of the bud with a meter stick.
3. Determine root collar diameter with a caliper at 1 cm above the
original root collar.
4. Separate each seedling into foliage, stem, and root components.
5. Determine oven dry weight (70°C) of each component.
6. Develop equations relating seedling height and diameter to
component dry weights.
7.3.2.2. INITIAL CHAMBER SEEDLING MEASUREMENTS
° Just prior to beginning treatment exposures, height and root collar
diameter of all seedlings should be measured.
7.3.2.3. END OF YEAR 1 MEASUREMENTS
1. Select 10 trees/family/treatment for destructive harvesting.
2. Divide the growth measurement trees into stems and foliage. Note:
No root biomass measures are required.
3. Randomly select a subsample of foliage from each family and
determine the specific leaf area (cm /g) as outlined in the SOP
for Measurement of Seedling Leaf Area (Section 6.3.2.).
4. Determine oven dry weight of the stem and foliage components.
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5. Determine the relationship between stem and leaf biomass and
measured diameter and height.
6. Estimate total leaf area from specific leaf area measurement and
leaf biomass measures.
7. Measure height and diameter of all seedlings remaining in the
chamber.
8. Estimate stem and leaf biomass of the seedlings that are to remain
in the study for year 2.
7.3.2.4. END OF EXPERIMENT GROWTH MEASURES
1. Measure height and diameter of all seedlings.
2. Clip all seedlings at the root collar.
3. Separate into foliage and stems.
4. Select a subsample of foliage and determine specified leaf area
for each family. See SOP for Measurement of Seedling Leaf Area
(Section 6.3.2.).
5. No root biomass estimates are required.
7.4. PREVENTIVE MAINTENANCE
° Keep caliper clean and oiled.
° Keep meter sticks in a container so they do not get damaged and
become difficult to read.
7.5. CALIBRATION PROCEDURES
1. At the beginning and end of a measurement period, the caliper
should be checked against a calibration gauge of known width.
2. After each reading, the caliper should be checked to ensure it
reads zero when the jaws are fully closed. If it does not, clean
the jaws if they have resin on them and adjust the caliper to read
zero.
3. Determine caliper accuracy and precision by measuring the
calibration gauges 5 times at the beginning and end of a
measurement day.
4. Analytical balances should be calibrated at the beginning and end
of each measurement session using a set of standard weights which
cover the range of measurements to be made. Readings should be
within 1% of the known weight readings. Analytical balances
should be spot checked using a single calibration weight that is
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near the mid point range of the samples being measured. If a spot
check deviates more than 1X from the known value, the analytical
balance should be checked for cleanness. If cleaning does not
help, the instrument should not be used until it can be
recalibrated.
7.6. CALCULATIONS/UNITS
6 Relative growth rate calculations should follow the procedures
described by Evans (1972).
0 Seedling biomass estimates from diameter and height measures can be
estimated using the procedures described by several authors (see
Section 7.8. References).
7.6.1. UNITS
Root collar diameter .mm-nearest tenth mm
Height cm-nearest half cm
Weight. mg-nearest mg
2 -1
Specific leaf weight cm mg
7.6.2. RECORDING FORMAT
Date 0-365
Time Standard Military
Treatment Number
Family Numeric code
Tree Number
Height cm-nearest half cm
Diameter mm-nearest tenth mm
Weight-foliage mg-nearest mg
Weight-shoot mg-nearest rag
Weight-root mg-nearest mg
7.7. ERROR ALLOVANCEF AND DATA QUALITY
Errors in growth measurements can result from errors in the
measurement of height, diameter, or weight. Height can be measured to the
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nearest 0.5 cm. Diameter can be measured to the nearest .01 mm. Due to
variability in stem dimension, diameter estimates made with a single stem
measurement will be only accurate to the nearest 0.1 mm.
Weight measurements will be made to the nearest \X of actual weight.
7.7.1. DATA QUALITY OBJECTIVES
Variable
Reporting
Units
Repeated Measurement
Error at
Lower Limit Upper Limit
Measurement
Accuracy
Tolerance
Seedling Height
0.5 cm
+
2X
+
2%
2%
Sapling Height
0.1 m
+
5%
+
5X
5X
Diameter
mm
+
5%
+
5%
5%
Plant Dry Weight
mg
+
1%
+
1%
2%
Root Weight
mg
+
1%
+
IX
2X
Stem Weight
mg
+
IX
+
IX
2X
Needle Weight
mg
+
U
+
n
2X
7.8. REFERENCES
Edwards, M.B. and H. McNab. 1979. Biomass prediction for young southern
pines. J. Forestry. May:291-292.
Evans, G.C. 1972. The Quantitative Analysis of Plant Growth. University
of Calif. Press, Berkeley and Los Angeles. 734 pp.
Haines, S.G. and C.B. Davey. 1979. Biomass response of loblolly pine to
selected cultural treatments. Soil. Sci. Soc. Am. J. 43:1034-1038.
Hatchell, G.E., C.R. Berry, and H.D. Muse. 1985. Non-destructive indices
related to aboveground biomass of young loblolly and sandpines on
ectomycorrhizal and fertilizer plots. For. Sci.
31:419-427.
Hunt, R. 1982. Plant Growth Curves. University Park Press, Baltimore.
247 pp.
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Ruehle, J.L., D.H. Marx, and H.D. Muse. 1984. Calculated non-destructive
indices of growth responses of young pine seedlings. For. Sci. 30(2):
469-474.
Taras, M.A. 1980. Aboveground biomass of Choctahatchee sand pine in
Northwest Florida. USDA For. Ser. Res. Pap. SE-210, 23 p.
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8. STANDARD OPERATING PROCEDURE FOR DETERMINATION OF
FOLIAR INJURY TO SEEDLINGS AND SAPLINGS
8.1. SCOPE AND PURPOSE
The assessment and description of foliar injury under controlled
conditions such as growth chamber and open-top chamber studies can be
useful in (1) determining how much functional foliage has been reduced by
exposure to pollutants and (2) developing guides for identifying observed
field damage. The proposed system will attempt to describe the extent of
damage as well as the cause of damage.
8.2. MATERIALS AND SUPPLIES
Illustrations of foliage having damage in 10% area intervals for the
species being studied should be developed to serve as guides for
estimating foliage damage.
8.3. PROCEDURES
8.3.1. SYSTEM FOR FOLIAGE DAMAGE ESTIMATES
The following system should be used to make estimates of visible foliage
damage.
Extent of
Vigor Class Damage Types of Damage
0 None
1 <102
2 >10* <202
3 >20% <30%
4 >30% <40%
5 >40% <50%
6 >50% <60%
7 >60% <70%
8 >70% <80%
9 >80% <90%
10 >90%
Types of damage could be chlorosis, necrosis, desiccation, or insects.
8.3.2. STEPS IN DETERMINING FOLIAR DAMAGE LEVEL
1. Use random procedures to select foliage to be assessed for damage.
2. Compare sample foliage with a set of standards having a known
percent of area randomly shaded.
3. Visually estimate the extent of foliar damage using the standard
guide.
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4. Code the type or types of damage that are visible.
5. Note: A designated person should make all the estimates if
possible. The person should coordinate and train all other
individuals estimating visual injury for that species.
8.4. PREVENTIVE MAINTENANCE
N/A
8.5. CALIBRATION PROCEDURES
N/A
8.6. CALCULATIONS/UNITS
Damage will be expressed in percent. The amount of damaged area for
the whole seedling can be estimated from the randomly selected damage
assessed foliage.
8.7. ERROR ALLOWANCE AND DATA QUALITY
It is unlikely that the total damage area can be estimated more
accurately than +20% except when damage approaches 100% or zero.
8.8. REFERENCES
N/A
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9. STANDARD OPERATING PROCEDURE FOR MYCORRHIZAL ASSESSMENTS
9.1. SCOPE AND PURPOSE
This SOP will outline the procedures available for quantifying
ectomycorrhizae on seedling roots for seedlings grown in containers for up
to one year. Due to sampling complications where multiple families are
grown in open-top chambers for 2-3 years, no procedures are being
recommended in this SOP for conducting mycorrhizal assessments in open-top
chamber studies.
9.2. MATERIALS AND SUPPLIES
° Water source for removing potting media
° Microscope
° Soil sieves or screens
0 Ruler
° Analytical balance
0 Defloculation agents
0 Plant material: The entire root system should comprise the sample
for seedlings grown in containers.
9.3. PROCEDURES
9.3.1. SAMPLE PREPARATION
Sample collection and preparation should follow the procedures
outlined by Grand and Harvey (1982). The number of plants that has to be
sampled cannot be specified for all studies. It will depend on the
variability in mycorrhizal infection in the study being conducted. In
studies that are inoculated in containers, usually a minimum of ten plants
per replication and 4-5 replications are required to show significant
differences at the .95 significance level.
9.3.2. MEASUREMENT - COUNTS
The method used to quantify the level of mycorrhizal infection will
depend on the study objectives, the size of the seedling, and the number
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of seedlings that have to be assessed. The methods that have been used
are:
° Direct counts of the entire root system (this is suitable for small
numbers of seedlings),
0 Direct counts of randomly selected roots (this procedure permits
assessment of more seedlings), and
° Counts of ectomycorrhizal tips (this procedure counts tips of live
and/or dead mycorrhizae. It should be the preferred method if the
study objective is to relate mycorrhizal activity to nutrient
cycling, turnover, etc).
These methods have been described in detail by Grand and Harvey
(1982).
Precision estimates can be made by having each person that is in
charge of making assessments count mycorrhizae on a set of ten, fifteen cm
root segments and repeat this five times.
9.4. PREVENTIVE MAINTENANCE
N/A
9.5. CALIBRATION PROCEDURES
There are no instruments to be calibrated. In nutrient cycling
studies, weight may have to be determined. Precision and accuracy and
calibration requirements outlined in Appendix B are applicable.
9.6. CALCULATION/UNITS
Results may be presented as
0 number of ectomycorrhizae per seedling.
0 number of ectomycorrhizae/unit length of root.
° percent of short roots with ectomycorrhizae.
° weight of ectomycorrhizal tips/unit area or weight of soil.
° weight of ectomycorrhizal tips/unit volume of soil.
9.7. ERROR ALLOWANCE AND DATA QUALITY
In container studies involving seedlings, the major sources of errors
will be:
° loss of fine roots in extracting the roots for mycorrhizae counts
58
-------
Section: 9
Revision: 0
Date: June 1986
Page: 3 of 3
for studies designed to assess mycorrhizae, using a 3:1
vermiculite:peat potting mix will facilitate root extraction), and
0 counting all forked short roots as mycorrhizal roots (as much as
15% of forked roots may not be mycorrhizal).
To minimize errors in mycorrhizal counts, it will be necessary that
certain people be assigned the task of making all mycorrhizal counts.
These people should attend a short course which covers how to
° recognize mycorrhizal roots,
° collect root samples,
0 store samples,
0 count mycorrhizal roots, and
° quantify mycorrhizal tips.
9.8. REFERENCES
Grand, L.E. and A.E. Harvey. 1982. Quantitative Measurement of
ectomycorrhizae on plant roots, pp. 157-164. In: Methods and
Principles of Mycorrhizal Research, N.C. Schenck, ed., Amer.
Phytopath. Soc., St. Paul.
Marx, D.H., C.E. Cordell, D.S. Kenney, J.G. Mexal, J.D. Artman, J.W.
Riffle and R.J. Molina. 1984. Commercial vegetative inoculum of
Pisolithus tinctorius and inoculation techniques for development of
ectomycorrhizae on bare-root seedlings. Forest Sci. Monograph #25.
59
-------
REFERENCES
Berry, W.L., P.A. Hammer, R.H. Hodgson, D.R. Krizek, R.W. Langhans, J.C.
McFarlane, D.P. Ormrod, H.A. Poole, and T.W. Tibbitts. 1977. Revised
guidelines for reporting studies in controlled environment chambers.
Hort. Science 12:309-310.
Burmann, F.J. and K.A. Rehme. 1978. Instrumentation, pp. 2-1 to 2-24, In:
Handbook of Methodology for the Assessment of Air Pollution Effects on
Vegetation, W.W. Heck, S.V. Krupa, and S.N. Linzon, eds., Air Pollution
Control Association, Pittsburgh.
Carlson, L.V. and W.D. Johnstone. 1979. Use of the rhizometer to estimate
foliar surface area. Can. For. Serv. Bi-monthly Res. Note 38:9.
Coombs, J., 0. Hall, S.P. Long, and J.M.O. Scurlock (eds.). 1985.
Techniques in Bioproductivity and Photosynthesis. Pergamon Press,
New York, 298.
de Cormis, L., J. Bonte, and A. Tisne. 1975. Technique experimentale
permettant l'etude de 1'incidence sur la vegetation d'une pollution par
le dioxyde de soufre appliquee en permanence et a dose subnecrotique.
Pollut. Atmos. 17:103-107.
Delta-T Devices, Ltd. Delta-T Devices: Scientific Instruments for Ecology.
16 pp. (Sole U.S. Distributor is Decagon Devices, Inc., NW 800 Fisk,
Pullman, Washington, 99163.)
Drew, A.P. and S.W. Running. 1975. A comparison of two techniques for
measuring surface area of conifer needles. For. Sci. 21: 231-232.
Edwards, M.B. and H. McNab. 1979. Biomass prediction for young southern
pines. J. Forestry. May:291-292.
Evans, G.C. 1972. The Quantitative Analysis of Plant Growth. University of
Calif. Press, Berkeley and Los Angeles. 734 pp.
Grand, L.E. and A.E. Harvey. 1982. Quantitative measurement of
ectomycorrhizae on plant roots, pp. 157-164. In: Methods and
Principles of Mycorrhizal Research, N.C. Schenck, ed., Amer. Phytopath.
Soc., St. Paul.
Greenwood, P., A. Breenhalgh, C. Baker, and M. Unsworth. 1982. A computer-
controlled system for exposing field crops to gaseous air pollutants.
Atmos. Environ. 16:2261-2266.
Haines, S.G. and C.B. Davey. 1979. Biomass response of loblolly pine to
selected cultural treatments. Soil. Sci. Soc. Am. J. 43:1034-1038.
Hatchell, G.E., C.R. Berry, and H.D. Muse. 1985. Non-destructive indicies
related to aboveground biomass of young loblolly and sandpines on
ectomycorrhizal and fertilizer plots. For. Sci. 31:419-427.
60
-------
Heagle, A.S., R.B. Philbeck, and W;W. Heck. 1973. An open-top chamber to
assess the impact of air pollution on plants. J. Environ. Qual. 2:365-
368.
Heitschmidt, R.K., V.K. Laurenroth, and J.L. Dodd. 1978. Effects of
controlled levels of sulphur dioxide on Western Wheatgrass in a
southeastern Montana grassland. J. Appl. Ecol. 15:859-868.
Hogsett, V.E., D.P. Ormrod, D. Olszyk, G.E. Taylor, Jr., and D.T. Tingey.
1986. . Air Pollution Exposure Systems and Experimental Protocols: A
Review and Evaluation of Performance. (In preparation).
Hunt, R. 19B2. Plant Growth Curves. University Park Press, Baltimore. 247
pp.
Johnson, J.D. 1984. A rapid technique for estimating total surface area of
pine needles. For. Sci. 30:913-921.
Jurgens, R.B. and R.C. Rhodes. 1976. Quality Assurance and Data
Validation for the St. Louis Regional Air Pollutant Study. In:
Proceedings of the conference on Environmental Modeling and Simulation,
U.R. Ott, ed. EPA 600/9-76-016.
Rats, G., C.R. Thompson, and W.C. Kuby. 1974. Improved ventilation of open
top greenhouses. J. Air Pollut. Contr. Assoc. 26:1089-1090.
Kramer, P.J. and T.T. Kozolwski. 1979. Physiology of woody plants.
Academic Press. New York. 811 pp.
Krizek, D.T. and J.C. McFarlane. 1983. Controlled-environment guidelines.
Hort. Science 18:662-664.
Kvet, J. and J. Marshall. 1971. Assessment of leaf area and other
assimilating plant surfaces, pp. 517-555. In: Plant Photosynthetic
Production - Manual of Methods, Z. Sestak, J. Catsky, and P.G. Jarvis,
eds. V. Junk Publications, The Hague.
Lee, J.J. and R.A. Lewis. 1978. Zonal air pollution system: Design and
performance. Ecological Research Series EPA 600/3-78-021, pp. 322-344,
U.S. Environmental Protection Agency, Corvallis, OR.
Lewin, K.F. and L.S. Evans. 1985. Design and operation of an experimental
system to determine the effects of rainfall acidity on vegetation.
Brookhaven National Laboratory Report 34649.
LI-C0R. 1979. LI-3000 Area Meter Instruction Manual. LI-COR, Inc.,
Lincoln, Nebraska. 28 pp.
LI-COR. 1984. Area Meters: LI-COR Instrumentation for Biological and
Environmental Sciences. LI-COR, Inc., Lincoln, Nebraska. 7 pp.
61
-------
Mandl, R.H., L.H. Weinstein, D.C. McCune, and M. Keveny. 1973. A
cylindrical open top field chamber for exposure of plants to air
pollutants in the field. J. Environ. Qual. 2:371-376.
Marx, D.H., C.E. Cordell, D.S. Kenney, J.G. Mexal, J.D. Artman, J.W. Riffle
and R.J. Molina. 1984. Commercial vegetative inoculum of Pisolithus
tinctorius and inoculation techniques for development of ectomycorrhizae
on bare-root seedlings. Forest Sci. Monograph #25.
McFarlane, J.C. 1981. Measurement and reporting guidelines for plant
growth chamber environments. Plant Science Bui. 27(2):9—11.
McLeod, J.A.R., J.E. Fackrell, and K. Alexander. 1985. Open-air fumigation
of field crops: Criteria and design for a new experimental system.
Atmos. Environ. 19:1639-1649.
Miller, J.E., D.G. Sprugel, R.N. Muller, H.J. Smith, and P.B. Xerikos.
1980. Open-air fumigation system for investigating sulfur dioxide
effects on crops. Phytopathology 70:1124-1128.
Mooi, I.J. and A.J.A. van der Zalm. 1985a. Research on the effects of
higher than ambient concentrations of SOj and N0„ on vegetation under
semi-natural conditions: The developing and testing of a field
fumigation system; process description. First Interim Report, January-
December 1983.
Mooi, I.J. and A.J.A. van der Zalm. 1985b. Research on the effects of
higher than ambient concentrations of SOj and N0„ in vegetation under
semi-natural conditions: The developing and testing of a field
fumigation system; Execution. Interim Report, January-December, 1984.
Moskowitz, P.D., V.H. Medeiros, N.L. Oden, H.C. Thode, Jr., E.A. Coveney,
J.S. Jacobson, R.E. Rosenthal, L.S. Evans, K.F. Lewin, F.L. Allen.
1985. Effects of Acid Deposition on Agricultural Production, BNL51889,
Brookhaven National Laboratory, Upton, New York.
Muller, R.N., J.E. Miller, and D.G. Sprugel. 1979. Photosynthetic response
of field-grown soybeans to fumigations with sulphur dioxide. J. Appl.
Ecol. 16:567-576.
Olszyk, D.M., T.M. Tibbitts, and V.M. Hertsberg. 1980. Environment in
open-top field chambers for air pollution studies. J. Environ. Qual.
9:610-615.
Ruehle, J.L., D.H. Marx, and H.D. Muse. 1984. Calculated non-destructive
indices of growth responses of young pine seedlings. For. Sci. 30(2):
469-474.
Sestak, Z., J. Catsky, and P.G. Jarvis. 1971. Plant Photosynthetic
Production - Manual of Methods. W. Junk Publications, The Hague.
Shelton, M.G. and G.L. Switzer. 1984. Variation in the surface area
relationships of loblolly pine fascicles. For. Sci. 30:355-363.
62
-------
Shriner, D.S., J.V. Johnston, Jr., G.E. Taylor, Jr., R.J. Luxmoore, R.K.
McConathy, S.B. McLaughlin, A.S. Heagle, R.J. Norby, B.K. Takemoto, D.T.
DuBay, C.H. Abner, D.D. Richter. 1985. Acidic deposition: Effects on
agricultural crops. Final Report to the Electric Power Research
Institute (Project 1908-2).
Spomer, L.A. 1981. Guidelines for measuring and reporting environmental
factors in growth chambers. Agron. J. 73:376-378.
Summary of Performance Test Results and Comparative Data for Designated
Equivalent Methods for SC^. 1979. QAD/M-79.12. (Complete reference
unavailable.)
Taras, M.A. 1980. Aboveground biomass of Choctahatchee sand pine in
Northwest Florida. USDA For. Ser. Res. Pap. SE-210, 23 p.
Thompson, F.B. and L. Leyton. 1971. Method for measuring the leaf surface
area of complex ahoots. Nature 229:572.
U.S. Environmental Protection Agency. 1978. Screening Procedures for
Ambient Air Quality Data. EPA-450/2-78-037 (OAQPS 1.2-092).
63
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APPENDICES
64
-------
APPENDIX A
List of Attendees
Exposure Systems and Physiological
Measurements Workshop
QA Methods Manual Development
Raleigh, NC
11-12 March 1986
Ruth Alscher
Boyce Thompson Institute
Cornell University
Tower Road
Ithaca, NY 14853
(607) 257-2030
Charles F. Baes III
Oak Ridge National Laboratory
Environmental Sciences Division
P. 0. Box X
Oak Ridge, TN 37830
(615) 576-2137
Jim Bailey
Texas A&M University
Forest Genetics Laboratory
College Station, TX 77843
(409) 845-1325
John Duff Bailey
U.S. EPA
200 SV 35th Street
Corvallis, OR 97333
(503) 757-4324
Roger Blair
Quality Assurance Director
Corvallis Environmental Research
Laboratory
200 SW 35th Street
Corvallis, OR 97333
Boris Chevone
Assistant Professor
VPI&SU
Dept. Plant Path., Physiology
Blacksburg, VA 24060
(703) 961-7871
Phillip Dougherty
Forest Resources
Univ. of Georgia
Athens, GA 30602
(404) 452-6566
Gwen Eason
N.C. State University
Plant Path. Dept.
Raleigh, NC 27695
(919) 737-3962
Lance Evans
Manhattan College
Department of Biology
Laboratory of Plant Morphogenesis
Riverdale, NY 10471
(516) 282-3458
Richard Flagler
N.C. State University
Crop Science Dept.
Raleigh, NC 27695
(919) 737-3575
Walter W. Heck
Air Quality Research Programs
1509 Varsity Drive
Raleigh, NC 27606
(919) 737-3311
Jack Jorgensen
USFS
Soil Science
P. 0. Box 12254
RTP, NC 27709
(919) 541-4217
Kimberly Joyner
NCSU Acid Deposition Program
1509 Varsity Drive
Raleigh, NC 27606
(919) 737-3520
65
-------
Lance W. Kress
Argonne National Laboratory
Bldg. ER 203
9700 So. Cass Avenue
Argonne, IL 60439
(312) 972-4212
Janet McFayden
NCSU Acid Deposition Program
1509 Varsity Drive
Raleigh, NC 27606
(919) 737-3520
Susan Medlarz
Northeastern Forest Exp. Stat.
USDA Forest Service
370 Reed Rd.
Broomall, PA 19008
(215) 461-3014
David M. Olszyk
Statewide Air Pollution
Research Center
900 University Avenue
University of California
Riverside, CA 92521
(714) 787-4716
Richard A. Reinert
Air Quality Research Program
North Carolina State University
1509 Varsity Drive
Raleigh, NC 27606
(919) 737-3962
Curt Richardson
School of Forestry & Environ.
Studies
004A Bio Science Building
717 Anderson Street
Duke University
Durham, NC 27706
(919) 684-2619
(919) 684-2421
Michele Schoeneberger
USDA Forest Service
Box 12254
3041 Cornwallis Road
Research Triangle Park, NC 27709
(919) 541-4213
John R. Seiler
VPI & SU
Forestry Department
228 Cheatham Hall
Blacksburg, VA 24061
(703) 961-4855
Steven Shafer
N.C. State University
Air Quality Program
1509 Varsity Drive
Raleigh, NC 27606
(919) 737-2142
Art Wiselogel
Research Associate
Forest Science Dept.
Texas A&M
College Station, TX
(409) 845-5033
66
-------
Table 1. Table of messurementa, SOP reference* end accuracy & precision levels required for aeasures of net carbon exchange (NCX), water vapor exchange, xylem pressure potential
and the required associated environment & plant varlablea.
Measurement/Method
Net Carbon Exchange
(NCC)
Standard Operation
Procedure Citations
LI 6000 - Manual
ADC-LCA2 - Manual
Additional Operation
\ Precision % Accuracy Inatruction/Requirements
• Boundary layer resist-
ance S .25 a/en
• Cuvette light transait-
tance 85\ of a rev
chaaber.
Callb Frequency Spot Qieck frequency
Cautions: Consents
00j Analyser &
Chaaber a
Sestak, Cat8ky &
Jarvla - Plant
Photosynthesis Pro-
duction Manual of
Methoda
< 1*
variation
± 5 ppm
Leaf temperature ele-
vation 3 2aC due to
enclosure of foliage
within the cuvette.
Draw down of CO. con-
centration should be 2
IS ppa for a closed
systea aeaaurement of
NCE at ambient CO.
levels.
Span - Dally
&
Zero - Dally
Zero - hourly
On
*-4
Leaf & Air Te
perate
Thermistors, thermo-
couples
± I.0*C ± 1.0*C • Conifer leaf teapera-
ture to be taken aa air
teaperature taken with
shielded thermocouple
mounted In the chaaber.
3 months
Compare chaaber &
unit theralators
dally.
Avoid chaaber
thermistor being
exposed to direct
sunlight.
Avoid long measure-
ments which lead to
high foliage tem-
perature buildups
under high teapera-
ture & high radia-
tion loading. Tea-
perature should be
held with 2°C of
aablent.
~tl
1
M
X
C0
Photosynthetl-
cally Active
Radiation
Quantu
t 2k
± 5%
All measurements to be
taken with aenaor held
level.
Note deviatlona from
full sunlight. Pull
sunlight will be
close to 2000
liaoles/m'/s. If
deviation froa full
sunlight 1s greater
than 400 itBoles/a'/a
on a clear day at
solar noon during the
aumaer period. Check
the sensor against
another aenaor that
has a more recent
calibration.
Relative humid-
ity
Thin layer capacitor
type sensor
± 2\
i 5*
Weekly
When operating under
hualdltlea > 90\ with
strong oxidants in
the atmosphere, call
brat Ion should be
checked twice weekly
until It la estab-
lished whether rapid
deterioration of the
sensor is occurring.
If water condenses on
sensor, let dry & re-
calibrate before re-
iisIdr.
-------
Table 1. Continued
*tl 3000 * 5\ * 5\
(Projected
*L1 3100 - Manual Area)
Delta-T - Manual
"Volume Displacement t 5H t 5\
Method* (Total
Surface
Area)
Voluae Displacement
00
• Shelton, H.G. &
G.L. Swltier
(1980 - Appendix
• McLaughlin. S.B. 6
H.A.I. Madgalck
(1966)
• Gurumurtl, K. L
V.K.S. Rirastara
(1982)
• Jon D. Johnaon
(1984) - Appendix
A Rapid Technique
for Eatimatfng
Total Surface Area
of Pine Needle*
Leaf Weight Weight Measurement IX ± 1.5*
Section
NCLAN Q/A Plan for
Biological Maaaure-
menta
Stooatal Conductance LI-1600 - Manual
Stoaatal Realatance Delta-T - Manual
Xylem preaaure PMS & Soil Molature * 0.1 MPa No check
potential Teat Owner'a Manual Eatabltah pre*
cialon esti-
mate acroaa
the range of
xylea preaaure
potentlala
that are expe-
rienced In the
study.
Recheck calibration No natter which pro-
after every 50 earn- Jected area aethod la
plea. selected for use with
conlfera that do not
have flat needles, It
will be necessary to
develop a calibration
curve relating pro-
jected area to total
surface area. When
the atudy Involves
both prlbary & sec-
ondary needles, sepa-
rate calibration
curvea will be re-
quired for each
needle type.
Non~requlred
A calibration la
to be ude at the
beginning and end
of each measure-
ment aeaslon with
a calibration
plate that has a
similar area &
configuration of
the Individual
tissue aaBple be-
ing measured.
Same aa for NCE measure-
ment a described above,
except for references to
CO. condltlona.
Calibrate at the
beginning & end
of each weigh"
lng aeaaion.
If weighing
aesslon is
greater than k
houra, check
every 4 houra.
Factory
Callb annually
Spot check a aid
point atandard
weight every 30
measurements.
aa for NCE above.
ft
a
Ou
None required
Check that pressure
needle goes to aero after
each XP determination.
-------
APPENDIX C
LI-6000 Portable Photosynthesis System
Service included vith 6000CAL Factory Calibration
Calibrations
1. Humidity Sensor*
2. C02 Analyzer re linearization* (7 gas concentrations at 3
temperatures; 18 linearization points are derived from a
computer program)
3. Flow meter relinearization (7 different flow rates at 3
temperatures)
4. Chamber temperature
5. Leaf temperature
6. Quantum sensor
* customer is informed of what the reading was when it was
received and what the new values are. This shows the amount of
calibration drift that occurred. Calibration certificate sent
with each instrument.
Testa
1. Check all 6000B Rechargeable Batteries that are returned:
a. Charge overnight
b. Timed discharge at 1 amp
c. Evaluate results
d. Charge overnight
e. Force charge for a period of time, dependent on step 3
evaluation
f. Timed discharge again
g. Repeat until 2 amp-hour reached or battery is not able to
be revived (generally 2 force charges is the maximum needed
to determine this).
h. Customer is informed of battery results and whether
replacements are needed.
2. Test pump (air flow rate).
3. Check and fix any air leaks in the system (inform customer of
leaks found).
4. Check accuracy and repeatability of C02 analyzer and flow
meter.
5. Check noise levels of C02 analyzer and flow meter.
6. 1/0 check (dump data to terminal or printer).
7. Test A/D, multiplexer accuracy/linearity.
8. Check chamber fan current drain; replace fan if excessive
(fan not included).
69
-------
Appendix C (continued)
General Maintenance
1. Replace Ni-Cad back-up batteries for RAM board if over one year old.
2. Replace air filters in LI-6050.
3. Check for loose nuts and screws.
4. Replace chamber pads as needed.
5. Clean chambers and instruments as needed.
6. Replace hoses as needed.
7. Connectors: Replace O-rings and grease as needed.
8. Install any upgrades that are included as no-cost upgrades.
9. Replace soda lime with new soda lime.
10. Replace 6000DP filter disk on desiccant tube as needed.
70
-------
APPENDIX D
COMPUTER CODES AND REPORTING SYMBOLS FOR CERTAIN MEASURED VARIABLES
Reporting
Variable Name
Code
Symbol
Leaf Conductance
LCOND
1/
gir
Leaf Resistance
LRES
ru
Canopy Conductance
CCOND
gcj
Transpiration
TRAN
Et
Photosynthetically Active Radiation
PAR
PAR
Air Temperature
ATEMP
T
a
Soil Temperature
STEMP
T
s
Relative Humidity
RHUM
RH
Leaf Temperature
LTEMP
T1
Vapor Pressure Deficit
VPD
VPD
Vapor Pressure Gradient
VPG
VPG
Soil Water Potential
SH20
T
s
Soil Water Content
SWATR
6
Xylem Pressure Potential
XPP
XPP
Specific Leaf Area
SLA
SLA
Leaf Surface Area
LAREA
A1
Leaf Area Index
LAI
LAI
Apparent Net Photosynthesis
NPS
A
n
Dark Respiration
DRESP
A
r
Ambient Carbon Dioxide
C02A
c
a
Internal Carbon Dioxide
C02I
c.
l
Photorespiration
PRESP
A
pr
1/ lj refers to species nts or molecule eg. G^^ = leaf
conductance to COj 2
71
-------
APPENDIX E
LI-1600 STEADY STATE POROMETER
1600CAL FACTORY CALIBRATION DESCRIPTION
Calibrations
1. Relative Humidity* (standard range 25-75%)
2. Mass Flow Meter*
3. Cuvette Temperature
4. Leaf Temperature
5. Quantum Sensor
* calibration certificate sent with each instrument includes readings taken before and after
calibration, indicating the amount of drift that has occurred.
Tests
1. Test pump (air flow rate)
2. Check for and repair any air leaks in the system. (Customer informed of leaks found.)
3. Check cassette and RS-232 interface operation.
4. Check HOLD switch for proper operation
5. Check "HUM SET" switch for proper operation.
6. Check power supplies for proper voltages. Calibrate if necessary.
7. Check flow controller for proper operation. Calibrate if necessary.
General Maintenance
1. Check for loose nuts and screws.
2. Clean instrument as needed.
3. Replace aperture pads as needed.
4. Replace hoses as needed.
5. Connectors: Replace and grease "O-Rings" on air line connectors as needed.
6. Check ribbon cables and connectors.
7. Visually check wiring connections.
8. Perform any routine upgrades.
9. Replace used dessicant with fresh dessicant.
10. Re-form leaf temperature thermocouple.
11. Check both fans for proper operation.
12. Recharge battery.
Replacement Parts Included
1. Urethane tubing for dessicant pack (2 pieces 2 1/2" long)
2. Spare aperture pads (3)
3. Desiccant
Price: U.S. $150 plus shipping. Other repairs needed are invoiced in addition to the 1600CAL
price.
A
If-CO#, me. l/COff, Uti.
-.j — — Box 4425 /Lincoln. Nebraaka 68504 USA
Phoo# (402) 467-3576 / TWX: 910-621-8116
¦A
-------
APPENDIX F
NATIONAL CROP LOSS ASSESSMENT NETWORK
QUALITY ASSURANCE PLAN
FOR BIOLOGICAL MEASUREMENTS
Approved:
^
NCLAN Bi
sj iU, f i
iologic/l Quality Assurance Leader
Approved:
Approved:
NCLAN, RMC, Chairman
Q^J ^ .0
i"
EPA Project Officer
Approved:
>¦ li, l.iUft-
n \
APEB, Chief
Approved:
Date:
Date: lo(J A C
Date: 7/ XI / fs
Date: / / B ^
Date:
CERL Quality Assurance Officer
All responsible personnel listed in the NCLAN Project will receive this
quality assurance plan for biological measurements plus any necessary
revisions.
73
-------
Appendix F (cont'd)
TABLE OF CONTENTS
Section Page
1.0. Introduction 1
2.0. Scope 2
3.0. QA Personnel and Responsibilities 3
4.0. QA Objectives and Procedures for Biological Measurements ...... 5
5.0. QA Data Handling Procedures
LIST OF FIGURES
Fig. 1. NCLAN Quality Assurance Weight Verification Check
Fig. 2. NCLAN Quality Assurance Calibration Checks for Weight Measurements
Fig. 3. NCLAN Quality Assurance Calibration Checks for Soil Moisture
Tensiometers
Fig. 4. Quality Assurance Calibration Checks for Area Measurements
Fig. 5. Data Flow and Verification for Manually Collected Biological
Data at ANL
Fig. 6. Data Flow and Verification for Manually Collected Biological
Data at UCR
Fig. 7. Data Flow and Verification for Manually Collected Biological
Data at BT1 and RAL
Fig. 8. Data Flow and Verification for Automatically Collected Biological
Data for All Locations
LIST OF TABLES
Table 1. Summary of NCLAN Biological Quality Assurance
1l
74
-------
Appendix F (cont'd)
1.0 Introduction
The objective of this quality assurance plan is to assure that biological
data collected for the National Crop Loss Assessment Network (NCLAN) is of
known quality, accuracy, and legally defensible. NCLAN objectives, experi-
mental designs, use of data, instrumentation QA plan, personnel, and locations
are found in the NCLAN Project Plans. This document specifies quality
assurance procedures required for biological measurements associated with
NCLAN research.
1
75
-------
Appendix F (cont'd)
2.0 Scope
Biological measurements made in NCLAN studies may include plant weights,
crop quality, soil moisture, and physiological measurements such as chloro-
phyll content, leaf water potential, leaf conductance (resistance) and/or
photosynthesis. The procedures outlined here are used to monitor these
variables and assure consistent biological quality assurance at each NCLAN
location.
Although the NCLAN program is designed to conduct research using a
common basic research protocol, because of regional, experimental, and/or crop
differences, the same measurements are not always made across sites or years.
Since NCLAN is a dynamic air pollution-crop research program, personnel
frequently develop and test new techniques, methods, and apparatus for making
biological measurements. When such procedures and/or apparatus are developed,
approved plans for assuring data quality will be added to this document before
procedures or equipment are used in the research.
2
76
-------
Appendix F (cont'd
3.0 QA Personnel and Responsibilities
1. NCLAN Research Management Committee (RMC)
The RMC manages all work conducted in NCLAN studies. NCLAN
quality assurance plans must be approved by this committee:
Walter W. Heck, Chairman, Agricultural Research Service, U.S.
Department of Agriculture, Raleigh, North Carolina
0. C. Taylor, Associate Chairman, University of California,
Riverside
Richard M. Adams, Oregon State University, Corvallis
Lance W. Kress, Argonne National Laboratory, Illinois
David T. Tingey, Environmental Research Laboratory, U.S. Environ-
mental Protection Agency, Corvallis, Oregon
Leonard H. Weinstein, Boyce Thompson Institute, Ithaca, New York
2. NCLAN Biological Quality Assurance Leader (NCLAN-BQAL)
The BQAL for all NCLAN locations is located at the Corvallis
EPA Laboratory. Responsibilities include (a) approving and
monitoring all QA plans; (b) compiling QA forms and preparing annual
evaluation reports. The NCLAN-BQAL (or designee) conducts at least
one site audit each year, contingent upon availability of travel
funds.
3. Site Leader
The Senior Scientist at each location is the BQAL unless other-
wise designated. Responsibilities include (a) ensuring that all QA
plans are followed at that site; (b) preparing and obtaining
approval for detailed QA plans when appropriate procedures are not
3
77
-------
Appendix F (cont'd)
included in the NCLAN QA document; (c) maintaining current samples
of approved verification, data sheets, and QA forms; (d) submitting
QA forms to the NCLAN BQAL by January 1; (e) reviewing notebooks for
completeness, calibration data, and preventive maintenance documen-
tation.
4
78
-------
Appendix F (cont'd)
4.0 QA Objectives and Procedures for Biological Measurements
A summary of biological variables to be measured, methods, equip-
ment, referenced standard operating procedures, minimum precision and
accuracy levels is shown in Table 1 in the Appendix.
For verification and tracking purposes, all samples and measurements
are coded so that the researcher can determine:
a. study number or species
b. harvest date and/or number
c. block or replicate number
d. treatment number or name
e. sample number
f. name of person(s) taking sample or measurement
1. Weight Measurements
The economically important portion of each crop is harvested at
maturity or, in case of fresh produce, when it is normally marketed.
Other plant parts also may be harvested for experimental purposes.
All crop material grown in the central 8-foot diameter of an
open-top chamber is normally harvested. Crop material from each
quadrant is harvested, yielding 4 samples per plot. Special studies
may require samples from smaller areas and in these experiments, the
sampling will be conducted according to the recommendations of the
NCLAN statistician.
The exact weight of samples is not required for NCLAN studies.
«
It is essential that all samples from a harvest or group be weighed
in the same manner. Use of procedures outlined in this section
5
79
-------
Appendix F (cont'd)
ensure that all samples are weighed within 1.5% of their true
weight.
Each NCLAN location maintains a set of standard weights
verified annually to 0.5% of another set of known standard weights.
Figure 1 illustrates a typical weight verification form. Scales or
balances are calibrated at the beginning and end of each weighing
period. For weighing sessions over 4 hours, a mid-session calibra-
tion check is conducted. Weights used for calibration will be
representative of the mid-range of samples to be weighed. To begin
or continue weighing each day, calibration checks must be within 1%
of the known value. Weighed samples are kept separate until
validated by a second calibration. If the mid-session or end-of-day
calibration is not within 1% of the known weight, all samples
weighed since the last valid calibration will be considered void.
They will be weighed again and considered, validated when a valid
before- and after-calibration check is conducted as stated above.
Figure 2 illustrates a typical calibration verification form.
When a calibration check deviates more than 1% of the known
value, the operator(s) first consult the operating manual to
identify possible problem areas. If unable to obtain a valid
calibration, the operator(s) notify the BQAL for the site to
initiate corrective action. This may indicate the need for a
maintenance check by a qualified equipment representative.
80
-------
Appendix F (cont'd)
NCLAN Quality Assurance Weight Verification Check
NCLAN Location:
Operator(s):
Brand Name and Serial #
% Difference = Working Wt - Standard Wt. 10Q
Standard Wt.
Weight Number
1
2 | 3
1
4 j 5
6 7
8 Comments
Date
j
!
i !
i i
Working i
I
|
1
i
i
Standard ! j
i
l
i
% Diff. |
i
Date | j
I
j
Working : !
: i
1
1
j
Standard | |
¦
1
!
f
% Diff. ,
1 i
Date . I
i !
i !
; ;
Working
; | i
i :
Standard \ j :
% Diff. !
i !
Date : |
:
1
1
•
Working j
!
1
1 1
1
Standard : j
I
i
: i
! I
% Diff. |
i
t
i
: 1
1 I
Figure 1
7
81
-------
Appendix F (cont'd)
NCLAN Quality Assurance Calibration Checks for Weight Measurements
NCLAN Location:
Study Name:
Operator(s):
Brand Name and Serial
Description of Samples:
^ _ Measured Wt. - Workinq Wt.
% Difference = r.— rr: a x 100
Working Wt.
i
Date
Start-Up Check
Mid-Session Check End of Day Check
Comments
Measured Working % Diff
Measured Working % Diff.
Measured Working % Diff.
!
i
!
i
i i
1
!
i i
* i
j_ i
i
!
i
i
i
i
i
i
i
i
i
!
i
i
i
1 i
i :
1 ! i
1
i
i
j
i
•
1 !
; 1 .
j
i
i
+
Figure 2
8
82
-------
Appendix F (cont'd)
Soil Moisture Measurements
Soil moisture measurements are made using the neutron probe
and/or tensiometers depending on equipment availability and/or site
soil characteristics.
In NCLAN studies, tensiometers are used to measure soil
moisture potential in atmospheres. Because of the nature of tensio-
meters, it is only possible to conduct before- and after-study
calibration checks. This is done by placing all probes at the same
depth in a soil of uniform moisture or in a container of water.
Readings are recorded by probe number (see form in Figure 3). A
soil sample may be taken at the same depth as the tensiometers to
obtain a direct measure of soil moisture (gravimetric) for compar-
ison.
A probe that does not read within 10% of the median level probe
will not be used in the study. Probes reading zero (defective) in
the pre-study check will not be used to calculate the median level
probe. Probes deviating from the 10% standard at the end-of-study
check will be recorded as suspect of invalid readings for the entire
study. Each site maintains a 10% extra supply of probes that meet
the 10% uniformity standard. These will replace defective probes
during the study If a probe is suspected of erroneous readings,
the operator first checks to see that the manufacturer's recommenda-
tions are being followed. If the probe is still suspect, the
operator notifies the site BQAL who will verify the need to pull the
probe, check it against the extras for verification, and if neces-
sary, replace it. See Table 1 for additional QA information.
-------
Appendix F (cont'd)
NCLAN Quality Assurance Calibration Checks for Soil Moisture Tensiometers
NCLAN Location:
Study Number:
Study Name:
Person(s) Calibrating:
Date Began Calibration: Date Ended Calibration:
v n Measured - Median
% Difference = M ..^ x 100
Median
Probe No. Measured Median # Diff.
i
!
I
1
1
1
1
I
1
i
Probe No. Measured Median # Diff.
1
I
1
I
Comments:
Figure 3
10
84
-------
Appendix F (cont'd)
The neutron probe is also used to measure percent soil
moisture. See Table 1 for QA information for this method.
3. Plant Health
Plant health is determined by daily visual examination of
treatment plots. Incidence of disease, pathogen, insect, or
nutrient deficiency are recorded in a logbook at each site and when
possible appropriate action is taken to correct the abnormality.
Disease severity is estimated by comparison with published
standards (C. James, A manual of assessment keys for plant diseases,
Canada Department of Agriculture, Publication No. 1458. 1971)
and/or estimated as a percentage of total leaf area affected (see
Table 1 for other QA information).
11
85
-------
Appendix F (cont'd)
Area Measurements
Leaf area measurement is taken with a meter that photo-
electronical ly detects and measures the amount a sample shades a
scanning light beam. See Table 1 for standard operating procedures.
Calibration checks are conducted at the beginning and end of
each measuring session. During lengthy (4 hours or more) sessions,
a mid-point calibration check is made. Test plates of known area
are available for operator calibration checks For each check, one
calibration test plate is measured. If the reading of the test
plate exceeds ± 5% of the known area, belt cleanliness, tracking,
and tension are checked. If the observed area of the test plate
still exceeds ± 5% of the known area, the site BQAL is contacted to
initiate corrective action. This can include adjusting the meter,
calibration settings, or calling a qualified equipment representa-
tive.
Once proper calibration is achieved, the observed mean area and
mean % difference of the test plate is recorded on the verification
form entitled "Quality Assurance Calibration Checks for Area
Measurements" (Figure 4).
Precision is determined by duplicating the measurement of every
30th sample, or a minimum of 3, whichever is larger. A notebook is
kept of all calibrations, precision tests, and adjustments.
-------
QUALITY ASSURANCE CALIBRATION CHECKS FOR AREA MEASUREMENTS
u v n:** Mean Observed Area - Known Area
. u .Mean % Difference = „ = x 100
Area Meter Known Area
Date
Project
Operator
Initials
Start-up comments regarding
condition of transparent belt
Start-up Check
End °£hi?ls1on
End of session
comments regardi
condition of are
meter. Operator
please initial.
Cleanli ness
Tracki ng
Tension
Mean
(cm2)
Known
(cm2)
Mean
Diff
Mean
(cm2)
Known
(cm2)
Mean
Diff.
Figure 4
13
-------
Appendix F (cont'd)
5. Physiological Measurements
Plant physiological measurements include:
a. leaf water potential
b. leaf conductance
c. leaf transpiration
d. photosynthesis
e. chlorophyll
These measurements are instantaneous readings of plant processes
which are not amenable to precision and accuracy statements and for
which no standards have been developed. It is not possible to
determine what level of process a particular plant should display at
any point in time. In making duplicate measurements of physio-
logical conditions, it is not possible to determine if the measure-
ments are being taken at the exact same point or if the same level
is being measured. Plant process levels vary from point to point
within a plant and can change rapidly over time.
Researchers at each NCLAN location follow and document (in
instrument logbooks) manufacturers' standardizations, checks, and
procedures (Table 1) to ensure proper operation and maintenance of
instruments used in making physiological measurements. All of the
above measurements may not be necessary in every study. Due to
funding restrictions, it may not always be possible to use the same
manufacturer and model of instrument for a particular process at all
sites.
14
88
-------
Appendix F (cont'd)
5.0 QA Data Handling Procedures
5.1 Field -- Collection Handling Procedures
For verification and tracking purposes, data is identified by:
1. location
2. year
3. crop(s)
4. pollutant(s)
Biological data collection for weights, soil moisture, photo-
synthesis (UCR only), leaf area, disease severity, leaf water
potential, and chlorophyll content are manually recorded on data
sheets. Data flow and verification checks for these measurements
and observations are shown in Figures 5, 6, and 7. Data sheets
and/or data notebook checks are made by the principal investigator
or BQAL at each site to ensure that manually collected data are
entered correctly. The last page of data on each harvest or group
is initialed by the principal investigator or the BQAL.
Data collected automatically for leaf conductance, leaf
transpiration, and photosynthesis, are read directly into a micro-
processor in the field. Data flow and verification checks for these
measurements are shown in Figure 8. Computer files for all
biological data (manually and automatically collected) are checked
by the principal investigator or the BQAL to ensure that the data
fields are complete and- the recorded values are reasonable before
data analysis is started.
All data sheets, punch cards, and data tapes are maintained for
at least 3 years by the BQAL at each location.
15
89
-------
Appendix F (cont'd)
Plant weights, leaf area, height, plant water potential, chlorophyll, injury,
soil moisture, etc.
Observational and Measured Data
v
Data Sheets
P. I. Checks for
Completeness and
Outliers
v
Computer Files
v
P.I. Checks
Storage (Tape, Cards,
Active Files, Paper Back-up)
Manual Analysis
P. I. Comparison
Analysis
Figure 5. Data Flow and Verification for Manually Collected Biological Data
at ANL
16
90
-------
Appendix F (cont'd)
Of iice +
P .! . checks
Observat ions
Data Sheets
Notebooks
Photocopj e
Keypunch
(compare?"
archived on Upe< prime t:— )prir,toutt'
run programs to perform.—.—.— ^printout
^desired analys&s
Figure 6. Data Flow and Verification for Manually Collected Biological Data
at UCR
17
91
-------
Appendix F (cont'd)
Manually Collected Biological Data
V
Data Sheets
Data Notebooks
v
P.I. Checks
V
Computer Files (verified
by double entry then compared)
P.I. Checks
v
Storage Tapes
for Analysis
Figure 7. Data Flow and Verification for Manually Collected Biological Data
at BTI and RAL
18
92
-------
Appendix F (cont'd)
Photosynthesis and Stomatal Conductance
Li Cor 1600 or
Li Cor 6000
Manual Record of Data Subset
Self-Contained Microprocessor
Checks
Comparison
Computer Files
v
Analysis
Storage
(tape)
Figure 8. Data Flow and Verification for Automatically Collected Biological
Data for All Locations
19
93
-------
Appendix F (cont'd)
Data analysis is conducted at each site using the Statistical
Analysis System (SAS) software package (ANOVA, regression, etc.) and the
Weibull function model as described by Heck et a^. , Environ. Sci.
Technol. Vol. 17, No. 12, pp. 572A-581A, 1983; and Rawlings and Cure,
Crop Science, In press, 1985.
Data Synthesis and Analysis
For verification and tracking purposes, all sets of NCLAN biological
data collected by the NCLAN data library at NCSU in Raleigh are identi-
fied using the following descriptive information.
General: Site, Year, Crop (common, Latin, cultivar)
Cultural: Dates (planting, emergence, start and end of exposure with
growth stage), soil type and name, row and plant spacing)
Exposure and Monitor Information: Pollutant and method of addition,
start and stop times each day, number of exposure days, description
of sequential monitoring.
20
94
-------
Appendix F (cont'd)
APPENDIX
21
95
-------
Appendix F (cont'd)
Table 1. Summary of NCLAN Biological Quality Assurance
Parameter/Method Sites
Standard Operating
Procedure Citation
% Precision
Requi red
% Accuracy
Requi red
Plant Weight
a. Balance
2. Soil Moisture
a. Depth
Neutron
Probe
b.
Surface
Neutron
Probe
Tensio-
meter
ALL
UCR,
RAL,
ANL
ANL
RAL,
BTI,
ANL
See weight measurements
section of this document
1. UCR -- Troxler 3220 Instruc-
tion Manual: 1981 Section
2-1 to 2-6 and 4-1 to 4-2.
2. RAL -- 503 DR Hydroprobe
Moisture Depth Gauge
Manual: May 9, 1984
Operation: Section 2, pp.
Calibration: Section 3, pp
3. ANL -- Troxler 3222 Instruc
tion Manual: 19 Section
2-4 to 2-6
Troxler 3411-B Series Surface
Moisture-Density Guage Manual
Operation, pp. 8-9
Irrometer Co. Reference Book
#24, pp. 2-14. Also see Soil
Moisture Measurements this
document
± 8
N/A
N/A
6-20
30-32
N/A
N/A
N/A
± 1.5
N/A
N/A
N/A
N/A
N/A
Plant Health
a. Visual ALL
Exami nation
Leaf Area
a. Area
Meter
ANL
BTI
RAL
Disease -- C. James, A manual
of assessment key for plant
diseases, Canada Dept. Agric.
Pub. No. 1458, 1971
Portable Licor LI-3000
Licor LI-3100 and 3000
Licor LI-3100
Leaf Water Potential
a. Pressure UCR, Plant Water Status Console
Bomb
ANL
BTI
b. Thermo- RAL
couple Psych
Leaf Conductance
a. Steady- UCR&
State BTI
Porometry
3000, Instruction Manual 1981,
pp. 5-9
PMS Pressure Bomb
Licor LI-1600, Instruction
Manual 1982, pp. 4-1 to 4-12
and 5-1 to 5-10
N/A
± 10
+ 10
+ 6
N/A
N/A
N/A
N/A
± 5
± 5
± 3
N/A
N/A
N/A
N/A
22
96
(continued)
-------
Appendix F (cont'd)
Table 1 (continued)
Parameter/Method Sites
Standard Operating
Procedure Citation
% Precision % Accuracy
Required Required
b. Rate of
Water
Accumulati on
7. Leaf Transpiration
a. Steady- UCR&
State BTI
Porometry
b. Vapor ANL
Pressure
Di fference
8. Photosynthesis
a. Isotope UCR
Porometry
b. C02 ANL
Depletion
.ANL Li-Cor LI-6000
Li-Cor LI-1600, Instruction
Manual 1982, pp. 4-1 to 4-12
and 5-1 to 5-10
LI-COR LI-6000, Instruction
Manual 19 , pp. 2-1 to 2-8
UCR mfg. Johnson et a}. (1979),
Photosynthetica 13:403
Li-Cor LI 6000
9. Chlorophyll
a.
ANL
BTI -- Arnon, D. J., 1949, Plant
Physiology 24:1-15
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
23
97
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