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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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114 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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117 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.