United States 

Department of 

Agriculture 

Forest Service 

Northeastern Forest 

Experiment Station 

Research Paper NE-641 

Quantifying Foliar Responses 

of White Ash to Ozone and 

Simulated Acid Precipitation: 

An Assessment Proposal for 

Forest Exposure Studies 

Leon S. Dochinger 

Keith F. Jensen 
Abstract 

Seedling populations represent an important linkage for assessing the effect of air 

pollution on forests. This study examines the foliar responses of white ash 

seedlings to ozone and acid precipitation as a means of identifying atmospheric 

deposition effects on forests. With white ash seedlings, regression analysis 

revealed significant increases.in,foliar stippling, with ri'sing ozone concentrations ,, 

from 7 to 15 pphm. NO stippling occurred in the absence of ozone or with acid rain 

treatment alone. However, exposure to ozone plus acid rain resulted in higher 

levels of foliar stipple as treatment concentrations increased. Until the inherent 

problems relating to the determination of the potential effects of air pollution on 

forests are resolved, the use of quantitative indices of injury to foliage and related 

growth impairments on susceptible and tolerant seedlings appears useful for 

assessing long-term ambient pollution effects on seedlings, trees, and forests. 

The Authors 

LEON S. DOCHINGER received a B.S. degree in 1950 and a Ph.D. degree in 

1956 from Rutgers University, and an M.S. degree in 1952 from Cornell 

University. In 1956, he joined the Central States Forest Experiment Station as a 

plant pathologist. From 1970 to 1985, he was the Project Leader of the 

Northeastern Forest Experiment Station's air pollution unit in Delaware, Ohio. He 

has recently conducted research on the effect of atmospheric deposition in 

eastern forests. 

KEITH F. JENSEN, plant physiologist, holds B.S. and Ph.D. degrees from Iowa 

State University. He joined the Northeastern Forest Experiment Station in 1963 

and is now at the Station's laboratory in Delaware, Ohio. He is the Project Leader 

of the air pollution unit in Delaware, Ohio, and is conducting research on the 

effects of atmospheric deposition on vegetation. 

Manuscript received for publication December 7, 1989. 

Northeastern Forest Experiment Station 

100 Matsonford Road, Radnor, PA 19087 

April, 1990 
Introduction 

Quantifying of genetic tolerance to atmospheric deposition 

is one of the more pressing problems for forest 

management. Although sufficient gene pools exist for 

selection and breeding, tree populations are continually 

affected by injurious concentrations of ambient pollution. 

High levels of indigenous pollutants have damaged world 

forests (Dochinger 1971) and long-term, ambient pollution 

over large expanses of forests in the United States has had 

adverse effects (Dochinger 1972; Miller and Millican 1971). 

lntraspecific variation in responses to SO2 and O3 among 

plant sources has been demonstrated with Norway spruce 

(Rohmeder and others 1962), eastern white pine (Dochinger 

and Seliskar 1970; Houston 1974), ponderosa pine (Karpen 

1970), American sycamore (Santamour 1969), red maple 

(Townsend and Dochinger 1974) and white ash (Jensen and 

Dochinger 1989). In addition, significant interclonal variation 

in tolerance to SO2 was demonstrated by hybrid poplars 

derived from the same parentage (Dochinger and others 

1972). This range of genetically controlled variability within 

species influences the feasibility of breeding or selecting 

pollutant-tolerant trees appear very promising. 

The objectives of this study were to evaluate (1) 

quantitatively, the foliar responses of white ash (Fraxinus 

americana) seedlings to ozone and acid precipitation and (2) 

the use of white ash seedlings as biological indicator plants 

for long-term assessment of their foliar-growth impairment 

by air pollution under ambient conditions. This information 

will aid in examining the effects of atmospheric deposition in 

order to measure the degree of stress in forests. 

Experimental Methods 

Plant Culture and Treatment Conditions 

In the spring of 1986, 200 one-year-old seedlings of white 

ash were chosen for uniformity and potted in a commercial 

soil mix (Metro-mix 500)' in 15-cm plastic pots. 

Approximately 14 days later, 63 plants that had broken 

dormancy were selected for study. Each plant was randomly 

assigned to one of nine treatment combinations: three 

ozone and three acid rain treatments. Each treatment 

combination was replicated 7 times with one seedling per 

replication. 

All seedlings were watered with 300 ml of tap water before 

their fumigation.After ozone (03) treatment for 2 days, the 

seedlings were placed in acid rain chambers where they 

received 1.25 cm of simulated rain. The rain treatment plus 

the initial watering were adequate for the water requirements 

'The use of trade, firm, or corporation names in this paper 

is for the information and convenience of the reader. Such 

use does not constitute an official endorsement or approval 

by the US. Department of Agriculture or Forest Service of 

any product or service to the exclusion of others that may 

be suitable. 

early in the study. Subsequently, with an increase in plant 

size, 600 ml was applied weekly. The nutrient needs of the 

seedlings were met initially, and again after 4 weeks, by the 

addition of 100 ml of soluble fertilizer (Peters 20-20-20 

General Purpose Fertilizer) to each pot. 

Atmospheric Deposition Treatments 

Atmospheric deposition treatments consisted of three 

simulated acid rains and three ozone fumigations. Solutions 

of pH 3.0, 3.5, and 4.2 constituted the acid rain portion of 

study treatment. Rain was prepared with an automatic 

titrator which added an acid solution of 2 parts 1M H2SO4 

and 1 part 1 M HN03 dropwise to deionized water containing 

background ions (Table 1) until the desired pH was attained. 

Composition of rain was prepared to simulate an average 

summer rainfall (Table 2). 

Table 1 .-Composition of simulated acid rain 

Acid rain mix before pH adjustmenta 

Compoundb uMlL 

MgS04 7H20 3 

CaS04 2H20 7 

(NH4)2S04 5 

NH4N03 6 

NH4CI 9 

KNOB 2 

NaN03 3 

apH adjustments made with an acid solution containing 2 

parts 1 M H2SO4 and 1 part 1 M HN03; pH 4.2 required .O1 

mllliter, pH 3.5 .ll mllliter and pH 3.0 54 mllliter 

bConstituents added to deionized water. 

Table 2.-Comparisons of ions present in average 

summer rainfall in Central Pennsylvania to those present 

in simulated acid rain (mean figures, 1977-81) 

Ion Summer rain Simulated 

1977-1 981 acid rain 

uEq/L 

so4 128 30a 

K 2 2 

No3 46 11 

Na 3 3 

NH4 25 25 

Ca 14 14 

CI 9 9 

Mg 6 6 - ppppp 

aBefore acid solution was added 

1 
The acid rains were applied once a week in plastic 

chambers equipped with 1.2 m-diameter revolving circular 

discs (2 rpm). Each application of 1.25 cm of rain lasted 

about 45 minutes. 

Fumigation with 0, 7, or 15 pphm O3 was applied in CSTR 

chambers (Heck and others 1978). The seedlings were 

exposed to ozone for 7 hours per day (0900 to 1600h) for 2 

consecutive days every week. Ozone was generated with a 

corona discharge-type ozone generator and verified with a 

thermoelectron ozone monitor. The latter was calibrated 

daily with a Monitor Labs 8500 calibrator. 

During the course of this 8-week study, plants were 

transferred from a maintenance greenhouse into the 

fumigation chambers in another greenhouse. They were 

fumigated on Monday and Tuesday. On Wednesday 

morning, the plants were removed from the fumigation 

chambers and placed in the rain chambers. After this 

treatment, the seedlings were returned to the maintenance 

green house. 

Data Collection and Analysis 

Every week, the leaflets of each leaf on all seedlings were 

rated for foliar injury. The classic symptom of ozone injury 

on broadleaf species is stipple. It is characterized on white 

ash by pin-size spots of purplish pigmentation on the 

adaxial surface of the leaves. As injury intensifies, the 

stipples usually coalesce and finally develop into massive 

tan-colored necrotic lesions,eventually resulting in premature 

leaf loss. The quantity of stipple produced on each leaflet 

was assigned a rating of 0, I, 5, 10, 25, 50, 75, 90, 95, 99, 

or 100 percent injury. To determine the overall stipple injury 

rating for each seedling, ratings for individual leaflets were 

tabulated and then divided by the total number of leaves 

evaluated. Foliar injury ratings were adjusted by arc sine 

transformation procedures before regression analysis. 

Results 

Visible foliar symptoms occurred on white ash plants 

exposed to ozone and pH 3.0 acid rain only. Acid rain injury 

was characterized by small (0.5 mm diameter), circular-like, 

necrotic lesions which, initially, appeared on the adaxial 

surfaces and occasionally became bifacial lesions on maturing 

foliage primarily. These foliar symptoms were noted during 

the 4th week of treatment and were not recorded since they 

occupied less than 1 percent of leaf surface. 

Ozone symptoms were discernible on older leaves usually 

after 3 weeks of fumigation, although foliar stipple was at 

times observed weekly on immature leaves. In the six 

ozone-acid rain treatments, stippling of white ash leaflets 

was observed on 63, 34, and 31 percent of the 7 pphm 

ozone, pH 3.0-, 3.5-, and 4.2-treated seedlings, respectively. 

For the 15 pphm ozone, pH 3.0-, 3.5-, and 4.2-treated 

plants, 86, 84, and 59 percent, respectively, were noted. 

An increasing ozone concentration from 7 to 15 pphm and 

rising acidity produced the most significant amount of 

stippling of white ash leaflets. Both pH 3.0 and 3.5 at 15 

pphm O3 accounted for the most foliar pigmentation across 

all other treatments (Fig. 1). In addition, a significant 

variation in leaflet stipples was observed after exposure to 

pH 3.0 in comparison to pH 4.2 rain at 7 pphm. Over the 

8-week period of study, a gradual increase was observed in 

the quantity of foliar stippling resulting from the various O3 

acid rain treatments. No foliar stippling was discernible in 

the non-ozone treatments. 

Discussion 

Seedlings are a critical link with future forests; therefore, 

they are an important population for exposure studies in the 

laboratory and in the forest environment. The primary 

purpose of this study was to evaluate the possibility of 

quantifying white ash foliar symptoms to injury from ozone 

and acid rain. The initial assessment of treatment effects on 

foliar symptoms indicated that a percentage rating of visible 

foliar injury is feasible and provides reliable, repeatable 

results. 

These findings may offer a possible basis for relating the 

percentage of visible foliar injury to growth impairments for 

laboratory and field assessments. Based on previous study 

with white ash, this species was reported to be very 

sensitive to O3 and acid rain. Regression analysis also 

accounted for significant variation in leaf dry weight, height, 

leaf area, and new-growth dry weight with an increase in 

ozone concentration from 7 to 15 pphm. In addition, all 

growth variables decreased with an increase in acidity from 

pH4.2 to 3.0. The decreases ranged from 6 percent for 

height to approximately 20 percent for leaf dry weight, leaf 

area, and new-growth dry weight (Jensen and Dochinger 

1989). 

Similar observations are reported by other researchers. 

Across all acid rain treatments, 9-week-old white ash 

biomass was suppressed by the application of ozone. 

Significant pollutant x pH interactions occurred in leaf area, 

total plant dry weight, and root-shoot ratios with decreasing 

pH and O3 treated seedlings (Chappelka and others 1988). 

For all treatments, increasing O3 concentrations resulted in 

significant linear decreases in root, leaf, and total dry 

weights; total stem; increased root-shoot ratio; and mean 

relative growth rates of the total plant, stem, root, and 

leaves in 5-week-old white ash. Visible symptoms from O3 

(15 pphm). simulated rain (pH 3.0), or O3 plus simulated 

acid rain (1 5 pphm and pH 3.0) were observed on 61, 61, 

and 54 percent of the seedlings in all treatments, 

respectively. The combined effect of O3 and acid rain 

resulted in significantly less root dry weight and root-shoot 

ratio at 5 pphm 03, and significantly less relative root 

growth rate and root-shoot ratio at 10 pphm O3 as rain pH 

decreased (Chappelka and Chevone 1986). 
OZONE (pphm) 

Figure 1 .-Effects of pHlozone on white ash foliar stippling. 

Various measurements may be used to examine genetic 

differences in air pollution on plants. Height growth, as a 

percentage of growth exhibited by treated or control 

progenies or clones of similar parentage, is a useful 

parameter for discerning tolerance or sensitivity to pollution 

(Dochinger and others 1972; Townsend and Dochinger 

1974). Tolerant lines generally show less growth depression 

than susceptible ones. Foliar injury, expressed as a 

percentage of each leaf injured or as a percentage of all 

leaves displaying symptoms, also may be used and is 

probably the most useful means for identifying air pollution 

effects on vegetation. 

A number of causal stress agents, including air pollution, 

cause visible symptoms on plant foliage. These injurious 

responses can aid in identifying cause and effect, including 

effects on yield. Consequently, until the problems 

concerning the assessment of the effects of atmospheric 

deposition on forests, are resolved, utilization of indices of 

injury to foliage and growth impairment on susceptible and 

tolerant seedlings will be useful for determining the nature 

and extent of long-term ambient pollution. 
Literature Cited 

Chappelka, A.H., Ill: Chevone, B.I. 1986. White ash seedling 

growth response to ozone and simulated acid rain. 

Canadian Journal of Forest Research. 16: 786-790. 

Chappelka, A.H, Ill; Chevone, B.I.; Burk, T.E. 1988. Growth 

responses of green and white ash seedlings to ozone, 

sulfur dioxide, and simulated acid rain. Forest 

Science. 4: 1016-1029. 

Dochinger, L.S. 1971. Symptoms of air pollution injury to 

broadleaf forest trees. In: Methods for identification and 

evaluation of air pollutants injurious to forests. 

Proceedings XV Congress of International Union 

Forestry Research Organization. IUFRO 92: 7-32. 

Dochinger, L.S. 1972. Impact of air pollution on forest 

tree plantings. In:The earth around us. Soil Conservation 

Society of America Proceedings. 27: 134-138. 

Dochinger, LS; Seliskar, C.E. 1970. Air pollution and the 

chlorotic dwarf disease of eastern white pine. Forest 

Science. 16: 46-55. 

Dochinger, L.S.; Townsend, A.M.; Seegrist, D.W.; Bender, 

F.W. 1972. Responses of hybrid poplar trees to sulfur 

dioxide fumigation. Journal of the Air Pollution Control 

Association. 22: 369-371. 

Heck, W.W.; Philbeck, R.B.; Dunning, J.A. 1978. A 

continuous stirred tank reactor (CSTR) system for 

exposing plants to gaseous air contaminants. US. 

Department of Agriculture. Tech. Bull. ARS-S-181. 

Houston, D.B. 1974. Response of selected Pinus strobus 

L. clones to fumigation with sulfur dioxide and ozone. 

Canadian Journal of Forest Research. 4: 65-68. 

Jensen, K.F.; Dochinger, L.S. 1989. Response of eastern 

hardwood species to ozone, sulfur dioxide, and acid 

precipitation. Journal of the Air Pollution Control 

Association. 39: 852-855. 

Karpen, D.H. 1970. Ozone and sulfur dioxide synergism: 

foliar injury to a ponderosa pine geographic race 

plantation in the Puget Sound Region. Plant Disease 

Reporter 54: 945-948. 

Miller, P.R.; Millican, A.A. 1971. Extent of oxidant air 

pollution damage to some pines. Plant Disease 

Reporter 55: 555-559. 

Rohrneder, E.W.; Merz, W.; von Schonborn, A. 1962. 

Breeding spruce and pine varieties relatively resistant 

against industrial exhaust fumes. 

Forstwissenschaftliches Centralblatt. 81: 321-332. 

Santamour, F.S., Jr. 1969. Air pollution studies on 

Plantanus and American elm seedlings. Plant Disease 

Reporter. 53: 482-484. 

Townsend, A.M.; Dochinger, L.S. 1974. Relationship of 

seed source and developmental stage to the ozone 

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Environment. 8: 957-964. 
Dochinger, L.S.; Jensen, K.F. 1990. Quantifying foliar responses of white 

ash to ozone and simulated acid precipitation: an assessment 

proposal for forest exposure studies. Res. Pap. NE-641. Radnor, PA: 

US. Department of Agriculture, Forest Service, Northeastern Forest 

Experiment Station. 4 p. 

Seedlings represent an important linkage for assessing the effect of air 

pollution on forests. This study examines the foliar responses of white ash 

seedlings to ozone and acid precipitation as a means of identifying 

atmospheric deposition effects on forests. 

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