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BIOMARKERS FOR DEFINING AIR POLLUTION EFFECTS
IN WESTERN CONIFEROUS FORESTS
Paul R. Miller
Pacific Southwest Forest and Range Experiment Station
4955 Canyon Crest Drive
Forest Service, U.S.D.A.
Riverside, California 92507
ABSTRACT
Biomarkers, as discussed in this paper, are considered to be
tissue level or whole plant level changes which can be directly
related to air pollution exposure. In western coniferous forests
the markers used most frequently include visible injury to foliage,
histological changes in needle tissue, elemental contents of leaf
tissue (sulfur, fluoride, etc.~. The pattern of markers is more
effective in indicating the cause of observed effects than a single
marker. The evaluation of air pollution effects is improved by
measuring the concentration of suspected pollutants, sampling
plants along gradients of decreasing pollutant deposition, and
controlled exposures of seedlings or small trees in enclosures
comparing ambient with carbon-filtered air. Land managers need
rapid methods of assessment of air pollution effects because large
numbers of trees are involved. At the same time more specific
and convenient markers are needed to distinguish the effects of
air pollution from other abiotic stress.
INTRODUCTION
Forest land managers need improved diagnostic procedures to differentiate air
pollution effects from the influences of other man-caused perturbations, the extremes of
weather, and the action of biotic diseases. Biomarkers have traditionally been used in
combinations because more than one line of evidence is usually needed! to confirm that an
air pollutant is the cause of an observed effect (Figure 1~. The purpose of this paper
is to provide a brief background on some of the methods frequently used in western
forest areas to assess air pollutant effects, and to consider some limitations of those
techniques involving biomarkers. Several aspects will be addressed: (1) observations
derived from inspection of foliage and whole tree crowns, and microscopic observation of
leaf tissue, (2) chemical analysis of foliage, and (3) manipulations that clarify the
response of biomarkers.
VISUAL DETECTION AND ESTIMATION OF EFFECTS
Visual detection and assessment of injury can be done with respect to individual
needles and leaves, or the entire crown. The particular symptoms caused by sulfur
dioxide, hydrogen fluoride, and ozone are well established for western tree species (Bega,
1978, Malhotra and Blauel, 1980~. Confirmation of the particular pollutant causing injury
is often easy because there is a known source nearby. When sources are not nearby and
pollutant concentrations are low or near natural background, particularly in the case of
111
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112
FOLIAGE
APPEARANCE
\
FOLIAGE \
PIGMENTS \
FOLIAGE \ \
CHEMICAL \ \
CONTENT — -
INCL. STABLE
ISOTOPE RATIOS
FINE R=T / IN WE WEST \
CONDITION ~ . , ~ ~
/ / ~ \ \ LANDSCAPE
/ / | \ DISTRIBUTION
/ / ~ \ OF INURED
TREE RING/ / . ~ \ BEES
ANALYSIS/ ~ \
PATTERN OF SPECIES AIR QUALITY
CITRON AFFECTED AND WEATHER
DE~RIO~TION DATA
FOLIAGE FOLIAGE
LONGEVllY HISTOLOGY
DO T EcnoN AND
DIAGNOSIS OF
AIR POLLUTANT
EFFtCTS ~ HEW
IN THE WEST
FOLIAGE
RESPONSE
it' / TOC~
\ / FILTERED AIR
~ /, /
RECIPROCAL
GRAFTING
Figure 1. Biomarkers and supporting information frequently used to assess air pollutant
effects in western coniferous forests.
ozone, it is much more difficult to find convincing evidence that observed symptoms are
caused by an air pollutant.
With ozone-injured trees the pattern of crown deterioration is a very important
visual clue. The abscission of older foliage is accelerated by ozone so that injured trees
have barren branches near the bole, and the lower branches appear to have fewer and
shorter needles. The crown deteriorates from the inside-out and the bottom-up. These
changes provide the essential markers for estimating change of tree condition over time.
Forest management agencies have employed these characteristics in repeated surveys of
conifer forests in the Sierra Nevada and other locations in the West (Prongs et al.,
1978).
A scoring method developed for ozone-injured pines in the San Bernardino National
Forest in southern California uses a combination of characteristics to derive a score for
individual trees (Miller, 1973~. These characteristics include the average number of
needle whorls retained in the upper and lower parts of the crown, a description of the
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113
condition and length of needles in each of the remaining whorls in both the upper and
lower crown, and an estimate of lower branch mortality. This information is gained by
a combination of hands-on and binocular inspection of the foliage and crown condition.
The sum derived from the whorl counts and weighted values for foliage condition, foliage
length and branch mortality was categorized as: 0, dead; 1-8 very severe; 9-14, severe;
15-21, moderate; 22-28, slight; 29-35, very slight; and 36 or above, no visible injury.
This method of evaluation was used successfully to describe condition of ponderosa pine
(Pinus ponderosa Laws.), and Jeffrey pine (P. Jeffreyi Grev. and Balf.) located at plots
along a gradient of decreasing ozone dose in the San Bernardino Mountains.
A supplementary method used mainly with sapling trees involved repeated counts
of surviving needles in single annual whorls observed for three years in succession,
starting during the year the needle whorl was formed. Rates of needle loss were
proportional to the ozone dose at each of 5 plot locations (Miller and Van Doren, 1981~.
Modified survey methods have been employed in the southern Sierra Nevada
mountains but in all cases the presence of the chlorotic mottle symptom was the key
variable. Pronos et al. (1978) established 242 ten-tree plots in the Sequoia National
Forest at locations where roads and trails intersected the 1220, 1525, 1830, 2135, and
2440-meter contour lines. Chlorotic mottle symptoms were evaluated on branches cut
from the lower crown with a pole pruner. The only datum recorded was the youngest
annual whorl with any evidence of chlorotic mottle. For example, a tree with
symptoms on the youngest (current year whorl of needles) was given a score of 0 and
described as very severe injury. At the opposite end of the scale, a tree with symptoms
only on the fifth-year whorl or older was given a score of 4 and described as having
very slight injury. Only rarely were trees encountered with more than 5 annual whorls
in the Sequoia National Forest and these were considered healthy. In 1977, 42 percent
of the plots had no injury symptoms, or only very slight injury symptoms, 52 percent
had slight injury, and 6 percent had moderate injury.
In a parallel series of observations in the Sequoia National Forest comparisons of
ozone-injury to 100 ponderosa pines distributed among four plots were made between
1975 and 1983 (Williams and Williams, 1986). The basis of comparison was the percentage
of trees having chlorotic mottle symptoms on each of the needle whorls present. The
incidence of chlorotic mottle generally increased between 1975 and 1983. Recent efforts
have been made to evaluate the various methods of gathering pine injury data (Muir and
Armentano, 1987). The main issues investigated were: 1) the comparison of binocular and
spotting scope observations of foliage with "hands-on" evaluations of foliage on branches
cut from the crown of the same trees; 2) the difference of foliage condition in the
upper and lower crown; 3) the bias introduced by different observers employing the same
method; and, 4) the within-tree sample size needed to reach a standard error that was
one-third of the specified detection limit (10 percent of the needle surface area occupied
by chlorotic mottle).
Observations of needle retention and chlorotic mottle using binoculars and spotting
scopes were weakly correlated with "hands-on" assessments of foliage from the upper
crown. Optical instruments underestimated both needle retention and chlorotic mottle in
the lower crown; however, after adjustment for observer bias, this method did meet the
criteria for accuracy and precision for needle retention but not chlorotic mottle. Needle
retention and chlorotic mottle did not differ significantly between upper and lower crown
but needle length was longer in the upper crown along with a larger amount of injury
caused by factors other than ozone. The differences between assessments made by two
"hands-on" observers was generally not significant. Observer differences with optical
instruments were substantial and varied more for upper crown observations than for
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lower crown observations. Five branches per tree constituted a sufficient sample size to
obtain significant differences at p = 0.05 for both "hands-on" assessments and
observations with optical instruments.
Questions pertaining to which data types may be best to construct an "index" of
tree injury, and what tree sample size may be required have also been investigated
(Duriscoe, 1988~. There are advantages to the use of an index comprised of several
symptoms or characteristics of injured plants (Muir and McCune, 1987~. An index allows
the simultaneous use of all of the observed variables during the data analysis phase.
According to Muir and McCune ( 1987) the ideal index uses quantitative information which
is equally weighted or carefully weighted. The elements should be additive; they should
have a minimum at zero and maximum at the highest possible level of injury, and have a
nearly linear relationship to dose.
The index proposed by Duriscoe (1988) contains four variables: chlorotic mottle
(percent of the surface area of each whorl), needle retention (percent of the fascicles
retained in each whorl), needle length (length in centimeters averaged at the tree level),
and crown density (live crown ratio representing the tree level). These variables are
weighted differently and added together to obtain a number between 0 and 100, where
larger numbers mean more injury. It was estimated that 15-20 trees per plot would be
required when employing the index, compared with 50 trees per plot if only one variable
was recorded per tree, namely, the percent of surface area of the three-year-old needle
whorl with chlorotic mottle. Finally, the use of the index decreases the amount of field
work and yet provides a more descriptive baseline estimate of tree condition for
comparison with future observations.
Rice et al., ( 1983) used visible symptoms on ponderosa pine foliage as a biomonitor
for changes in levels of ambient sulfur dioxide and fluoride at two sites in Montana.
Foliage was cut from plot trees annually and brought to the laboratory where
assessments of symptoms could be made under a dissecting microscope. The symptoms
evaluated were described as: needle tip burn, mottling, basal injury, needle surface area
injured, total necrosis, and healthy needles. Tissue content of sulfur and fluoride was
measured. These variables were analyzed separately and not combined into an index.
Over a five-year period most variables showed significant differences between tree
populations located at Colstrip (until recently pristine) and Billings (polluted by several
sources for 40 years).
In conclusion, visible injury ratings for foliage and tree crowns will continue to be
used on an operational basis to determine the spatial distribution and severity of air
pollutant injury to pines in California and other western states. Considerable time and
effort is required to make these surveys; therefore, it is important to continue to strive
toward a use of the same biomarkers and satisfactory sampling procedures so that data
taken by several agencies can be compared from year to year and from place to place.
The Air Quality Divison of the U. S. National Park Service is providing leadership in this
effort.
NEEDLE HISTOLOGY
The kinds of histological effects and their sequence of development depend mainly
on whether the injury is acute or chronic. One study of acute injury, typified by needle
tip necrosis of conifers, sampled the transition zone between necrotic and healthy tissue
to identify individual effects for each of several stress agents (Stewart et al., 1973~.
They examined the cellular changes in the transition zone tissue caused by natural
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senescence, sodium chloride, boron, moisture stress, winter injury, suffocation, hydrogen
fluoride, sulfur dioxide, ozone and combinations of hydrogen fluoride with sulfur dioxide
or ozone. It was concluded that mesophyll collapse, resin duct occlusion, transfusion
parenchyma hypertrophy and phloem abnormalities were commonly caused by several of
these stresses acting independently. Therefore, the application of needle histology to
diagnosis of acute injury has important limitations because cell and tissue responses in
the transition zone are the same for several stress agents.
The histological examination of needle tissue with chronic injury (usually no tip
necrosis) may offer brighter prospects in the case of ozone. Chronic ozone injury to
pine is commonly characterized as a destruction of the contents of the mesophyll or
plicate parenchyma cells. Affected cells are typically distributed randomly and are often
seen more frequently in the outer layers of the mesophyll but not necessarily in the
substomatal region (Evans and Miller, 1972~. Phloem abnormalities have not been
observed in pine needles with either acute or chronic ozone injury (Stewart et al., 1973,
Evans and Miller, 1972~.
A recent study of ponderosa pine employing both light and electron microscopy
confirmed the typical mesophyll damage by ozone and further characterized the
cytological changes associated with both ozone injury and natural senescence (Karenlampi,
1986~. Histological examination is definitely useful because the principal macroscopic
symptom of ozone injury (chlorotic mottle) may be induced by other stress agents.
In conclusion, histological examination will continue to be useful in the case of
ozone to explore for injury to additional tree species in which needle senescence may be
hastened without the obvious appearance of specific macroscopic symptoms. Recent
reports in the literature regarding tree declines in Europe suggest that histological
examination can be developed to finer levels of resolution. Histological changes will
continue to be the most effective when used in combination with other biomarkers of air
pollutant injury.
CHEMICAL ANALYSIS OF LEAF TISSUE
Tissue analysis for fluoride and sulfur have been an integral part of numerous
studies of pollutant effects. At the West Whitecourt study site in Alberta sulfate and
total sulfur content of the foliage of several tree species decreased with increasing
distance along an emission corridor extending from a gas desulfurization plant. Foliar
sulfate concentration was a better indicator of foliar loading than total sulfur (Legge et
al., 1981~. The stable isotope ratio derived from comparison of foliar sulfur, crustal
(soil) sulfur and the fossil sulfur emissions from the gas plant revealed a difference
sufficient to conclude that foliar sulfur accumulation was indeed from the fossil source.
Sulfate content was determined for pine needles collected at increasing intervals of
distance downwind from Bakersfield, California. There were many sources of sulfur
pollution in addition to the local petroleum industry. Therefore, the analysis of stable
sulfur isotope ratios of foliar sulfate and the petroleum in the local area did not
compare sufficiently to provide conclusive results (Taylor et al., 1986~.
The analysis of foliage for fluoride content along 10 sample transects extending
away from a fluoride source near Columbia Falls, Montana clearly identified the direction
of pollutant transport in terms of a gradient of decreasing foliar fluoride content
(Carlson and Dewey, 1971~. The measured values of tissue fluoride ranged from slightly
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116
more than 1000 ppm at one-half mile from the source to approximately 20 ppm at a
distance of ~ miles.
Both total sulfur and fluoride concentrations of ponderosa pine needles were used to
monitor the annual accumulations at tree plots near Billings and Colstrip, Montana. The
Billings plot had been influenced for 40 years by several pollutant sources. Both sulfur
and fluoride levels at Billings remained considerably higher than Colstrip during the
5-year sampling period, even though a new coal-burning power generating plant had
recently begun operation at Colstrip. These data provide a valuable background against
which to compare future sulfur and fluoride loading at Colstrip (Rice et al., 1983~.
The West Whitecourt study discovered an additional marker of sulfur pollution. A
dramatic increase in manganese content of foliage was observed with decreasing distance
to the source. This result coincided with a similar decrease in soil pH. Greater soil
acidity made manganese more available for uptake (Legge et al., 1981~.
In conclusion, analysis of tissue for suspected pollutants will continue to be very
useful. Recent improvements in the speed and versatility of laboratory analytical
equipment may encourage the investigation of the relationships of a whole suite of anions
and cations in symptomatic plant tissue.
MANIPULATIONS THAT CLARIFY THE RESPONSE OF BIOMARKERS
-
Temporary exclusion of foliage from pollutant exposure under natural conditions in
the forest provides an opportunity to observe the difference between the new healthy
foliage and that which continues to be exposed. Adjacent groups of 10 naturally
regenerated ponderosa pine saplings were observed for a 5-year period during which each
group received one of the following treatments: ambient air in a greenhouse-like
enclosure, carbon-filtered air inside a similar house, and ambient air outside of the
enclosures. The treatments were carried out during the May - September "smog" season
and greenhouse walls and roofs were removed during the winter. Chlorotic mottle
symptoms no longer appeared on the new foliage in the filtered air treatment, foliage
retention increased, and both height growth and diameter growth increased sharply
compared to both ambient air treatments. Foliage of an outside tree in the path of the
air exhausted from the filtered air chamber also ceased to exhibit symptoms while other
portions of the tree continued to show symptoms (Miller and Elderman, 1977~. These
results provided confirming evidence that ozone was the cause of foliage injury and tree
decline in the San Bernardino mountains.
The influence of ozone pollution on newly emerged seedlings of Giant Sequoia
(Sequoiadendron giganteum (Lindl.) Decne.) is currently being studied in an open-top
chamber experiment in Sequoia and Kings Canyon National Parks. The treatments include
ambient air inside and outside chambers, carbon filtered air, and proportional addition of
ozone at the level of 1.5 X ambient. The appearance of foliar symptoms in the ambient
and enhanced ozone treatments has provided the information needed to survey nearby
groves to determine the natural incidence of these symptoms. The results of one summer
show significantly higher shoot and root weights for seedlings in carbon filtered air
compared to the 1.5 X ambient treatment (Miller et al., 1988~. The particular advantage
gained from controlled exposure studies is that plant material is exposed to most of the
common abiotic and biotic stresses in the presence and absence of the pollutant.
Therefore, it is essential to locate such experiments in the natural habitat of the species
in question. In conclusion, the method of long-term exclusion of trees and seedlings
from polluted air in their natural environment may be at a threshhold of new discovery
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with the development and application of branch chambers. This technique will assist in
making comparative physiological measurements between seedlings and mature trees in
different pollution environments.
SUMMARY
Several observations can be made about past uses of biomarkers of pollutant injury
to trees that may make their use more effective in the future.
· Combinations of biomarkers must be chosen for each condition to be investigated
it is risky to rely on a single biomarker.
Sample size must be an important consideration and efforts should be made to
construct injury indices from groups of biomarkers.
The time required for gathering sufficient biomarker data may require months or
even years; it is desirable to identify combinations of biomarkers that yield
information more rapidly.
There is sufficient need for discovering and improving manipulations that
enhance the utility and specificity of biomarkers.
REFERENCES
Bega, R. V. 1978. Diseases of Pacific Coast Conifers. U. S. D. A., Forest Service,
Agric. Handbk. No. 521. 206 pp.
Carlson, C. E., and J. E. Dewey. 1971. Environmental pollution by fluorides in
Flathead National Forest and Glacier National Park. U.S.D.A. Forest Service,
Northern Region Headquarters, Missoula, Montana. 55 pp.
Duriscoe, D. M. 1988. Methods for sampling Pinus ponderosa and Pinus jeffreyi for the
evaluation of oxidant-induced foliar injury. Draft Final Report submitted to the
National Park Service, Air Quality Division, Denver. Eridanus Research Associates
and Holcomb Research Inst., Butler University, Indianapolis, IN. 39 pp.
Evans, L. S., and P. R. Miller. 1972. Ozone damage to ponderosa pine: a histological and
histochemical appraisal. Amer. J. Botany. 59:297-304.
Karenlampi, L. 1986. Relationships between macroscopic symptoms of injury and cell
structural changes in needles of ponderosa pine exposed to air pollution in
California. Ann. Bot. Fennici. 23:225-264.
Legge, A. H., D. R. Jaques, G. W. Harvey, H. R. Krouse, H. M. Brown, E. C. Rhodes, M.
Nosal, H. U. Schellhase, J. Mayo, A. P. Hartgerink, P. F. Lester, R. G. Amundsun
and R. B Walker. 1981. Sulphur gas emmissions in the boreal forest: the West
Whitecourt case study. Water, Air, Soil Pollut. 15:77-85.
Malhotra, S. S., and R. A. Blauel. 1980. Diagnosis of Air Pollutant and Natural Stress
Symptoms on Forest Vegetation in Western Canada. Info. Rept. NOR-X228, Canadian
Forestry Service, Edmonton, 84 pp.
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118
Miller, P. R. 1973. Oxidant-induced community change in a mixed conifer forest. In Air
Pollution Damage to Vegetation, Advances in Chem. Ser. No. 122: Amer. Chem. Soc.
pp 101-117.
Miller, P. R., and M. J. Elderman (Eds.~. 1977. Photochemical oxidant air pollutant
effects on a mixed conifer forest ecosystem. U. S. EPA-600/3-77- 104.
Environmental Research Laboratory, Corvallis, OR. 339 pp.
Miller, P. R., and R. E. Van Doren. 1981. Ponderosa and Jeffrey pine foliage retention
indicates ozone dose response. In Proc. Sym. Dynamics and Management of
Mediterranean-type Ecosystems, San Diego, CA. U.S.D.A. Forest Service, Berkeley,
CA. p. 621.
Miller, P. R., R. D. Wilborn, S. B. Schilling, and A. P. Gomez. 1988. Ozone injury to
important tree species of Sequoia and Kings Canyon National Parks. Draft Final
Report. Submitted to the National Park Service, Air Quality Division. Denver, CO.
53 pp.
Muir, P. S., and T. V. Armentano. 1987. Evaluating oxidant-induced injury to foliage of
Pinus ponderosa (West) and Pinus strobus (East). A comparison of methods. Interim
Report to the National Park Service, Air Quality Division, Denver, CO submitted by
Holcomb Research Inst., Butler University, Indianapolis, IN. 75pp.
Muir, P. S., and B. McCune. 1987. Index construction for foliar symptoms of air
pollution injury. Phytopathology 71:558-565.
Pronos, J., D. R. Vogler, and R. S. Smith, Jr. 1978. An evaluation of ozone injury to
pines in the southern Sierra Nevada. U.S.D.A. Forest Service. Report No. 78- 1.
Forest Insect and Disease Management, Region 5, San Francisco, CA. 17 pp.
Rice, P. M., R. A. Boldi, C. E. Carlson, P. C. Tourangeau, and C. C. Gordon. 1983.
Sensitivity of Pinus pond~erosa foliage to airborne phytotoxins: use in biomonitoring.
Can. J. For. Res. 13:1083-1091.
Stewart, D., M. Treshow, and F. M. Harner. 1973.
needle necrosis. Can. J. Bot. 51:983-988.
Pathological anatomy of conifer
Taylor, O. C., P. R. Miller, A. L. Page, and L. J. Lund. 1986. Effects of oxone and sulfur
dioxide mixtures on forest vegetation of the southern Sierra Nevada. Final Report.
Contract AO- 135-33. Submitted to the California Air Resources Board. Statewide
Air Pollution Research Center, University of California, Riverside, CA. 145 pp.
Williams, W. T., and J. A. Williams. 1986. Effects of oxidant air pollution on needle
health and annual-ring width in a ponderosa pine forest. Environ. Con. 13:229-234.
Representative terms from entire chapter:
air pollutant
