Site by Bennett Web & Design Co.
2009 SOUTHEASTERN NATURALIST 8(3):471–478
Ozone-Induced Stipple on Plants in the Cape Romain
National Wildlife Refuge, South Carolina
Donald D. Davis*
Abstract - During 1996–1998 and 2002–2003, field surveys were conducted within
the Cape Romain National Wildlife Refuge, located on the Atlantic coast of South
Carolina, to determine if refuge vegetation exhibited foliar symptoms (stipple)
induced by ambient ozone. Foliar stipple was observed on Rhus copallina (Winged
Sumac), Sapium sebiferum (Chinese Tallowtree), and Vitis sp. (wild grape). Grape
had the greatest frequency of stippled plants, followed by Winged Sumac and
Chinese Tallowtree. Chinese Tallowtree produced the most classic ozone-induced
stipple, and may have the most potential as a useful ozone-sensitive bioindicator.
Percentage of plants with ozone-induced symptoms varied among species and years.
The threshold value of CUM60 ambient ozone needed to induce symptoms on ozonesensitive
plants in the refuge was estimated to be approximately 9000 ppb-hrs.
The Cape Romain National Wildlife Refuge (Cape Romain NWR), administered
by the US Fish and Wildlife Service (USFWS), was established
in 1932 as a migratory bird refuge. The 25,993-ha refuge, part of the Carolinian-
South Atlantic Biosphere Reserve, is located along the Atlantic coast
of southeastern South Carolina and is managed to provide quality habitat
for a diversity of wildlife species. Most of the refuge is located on Bull Island,
an ancient barrier reef approximately 10 km long and 3 km wide, but
the refuge also encompasses several ha on the mainland. The Cape Romain
NWR contains barrier islands, salt marshes, coastal waterways, beaches,
fresh and brackish water impoundments, and maritime forests (unpublished
In 1975, Congress conferred wilderness status on 11,331 ha of the Cape
Romain NWR and designated the “Cape Romain Wilderness” as a Class I
air quality area, affording the area stringent protection under the Clean Air
Act as amended in 1977 (US Congress 1977). The act gave federal land
managers of Class I areas the responsibility to protect all air quality related
values (AQRVs), including vegetation, wildlife, water, soils, visibility, and
cultural resources. By federal law, AQRVs in Class I areas must be protected
from deterioration. However, despite special protection, Class I air
quality areas in eastern US have been adversely affected by ozone (Chappelka
et al. 2003; Davis 2007a,b; Davis and Orendovici 2006; Lefohn and
Manning 1995; Manning et al. 1996). In order to determine if deterioration
is occurring, baseline AQRVs must be established. Therefore, the overall
*Department of Plant Pathology and Penn State Institutes of Energy and the Environment,
The Pennsylvania State University, University Park, PA 16802; firstname.lastname@example.org.
472 Southeastern Naturalist Vol. 8, No. 3
objective of this survey was to determine if vegetation within the Cape Romain
NWR was being injured by ambient ozone, and if so, to what extent
it was occurring in the refuge. Specific objectives were to: 1) determine if
vegetation within the refuge was exhibiting foliar symptoms induced by
ambient ozone; 2) determine the incidence (percentage) of ozone-sensitive,
bioindicator plants exhibiting symptoms; and 3) if datasets were sufficient,
determine if incidence of ozone symptoms was related to species, ambient
ozone levels, and drought stress. This paper is the fourth in a series dealing
with ozone injury on vegetation within US national wildlife refuges (Davis
2007a,b; Davis and Orendovici 2006).
Stalter (1984) identified 268 species of native and introduced plants in
the Cape Romain NWR, and Helm et al. (1991) characterized the maritime
forest within the refuge. A species list of vegetation compiled by refuge personnel
was given to the author prior to the initial survey in 1996. From this
list, the author noted that the refuge contained several ozone-sensitive bioindicators
(later summarized in USDOI 2003), including Rhus copallina L.
(Winged Sumac) and Vitis spp. (wild grape). Bioindicators are broadleaved,
ozone-sensitive plants that respond to ambient ozone by producing characteristic,
diagnostic adaxial “stipple,” as first described by Richards et al.
(1958) and illustrated by Skelly (2000).
Ambient ozone is monitored within the Cape Romain NWR at EPA AIRS
Site #45-019-0046. The National Park Service, Denver, CO, provided the
EPA ozone data for several years prior to the initial 1996 survey. These datasets
revealed elevated levels of ambient ozone occurred within the refuge.
This information, along with the presence of potential ozone-sensitive bioindicators
in the refuge, provided the impetus for these surveys. Vegetation in
the Cape Romain NWR was surveyed during late summer in 1996–1998 and
2002–2003 on the dates listed in Table 1. Methods were similar to those used
by the author in other wildlife refuges (Davis 2007a,b; Davis and Orendovici
Prior to the initial survey in 1996, refuge maps were examined to identify
open areas with unrestricted air movement and sunlight, criteria for suitable
survey areas (Anderson et al. 1989). Potential locations were visited in early
1996. At this time, it was observed that wild grape and Winged Sumac occurred
frequently within the open areas along access roads and at the refuge
boat landing. It was also noted that Sapium sebiferum (L.) Roxb. (Chinese
Tallowtree) was very abundant in the refuge, especially in wet areas. Anderson
et al. (1986) had reported that Chinese Tallowtrees on the eastern
half of Bull Island exhibited symptoms resembling those caused by ozone.
Therefore, this species was added to the list as a possible bioindicator. Other
potential bioindicators were noted in the field, but they were generally few
in number and scattered.
2009 D.D. Davis 473
Based on these initial observations, the author selected those open survey
areas on Bull Island and within the mainland portion of the refuge that contained
the greatest numbers of Chinese Tallowtree, wild grape, and Winged
Sumac. At each survey area, the author counted the number of Chinese Tallowtree,
wild grape, and Winged Sumac plants, and recorded the number of
stippled plants within each species. Stipple was noted simply as present or
absent on individual bioindicator plants; severity (percent symptomatic leaf
tissue) of stipple was not evaluated. Percentage (incidence) of plants exhibiting
stipple within each of the three plant species was calculated.
Since species (USDOI 2003), ozone level (Hildebrand et al. 1996),
and drought stress (Eckert et al. 1999, Showman 1991, Yuska et al. 2003)
infl uence visible ozone-symptom expression, the author investigated the
relationship among these factors and incidence of stipple using binomial
logistic regression (Davis and Orendovici 2006, Minitab 2003). Bioindicator
species were numbered from 1–3. Annual ambient ozone levels were
expressed as CUM60 (ppb-hrs), the cumulative sum of all hourly ozone
concentrations ≥60 ppb, 24 hrs/day, during the time period from January 1 to
the first day of each year’s survey. Annual soil moisture as of July 31 of each
survey year was expressed as the Palmer Drought Severity Index (PDSI),
an approximation of drought stress based mainly on soil moisture and temperature
(Palmer 1965). PDSI was calculated using data for the southeastern
coast of South Carolina (USNCDC 2004). CUM60 ozone levels for April
through September of each survey year were graphed to illustrate temporal
ozone patterns during the growing season (Fig 1).
Results and Discussion
Ozone-induced stipple was observed on at least two of the bioindicator
species during each survey year, and all species exhibited classic adaxial
foliar injury (Richards et al. 1958, Skelly 2000). Relative prevalence of and
mean percentage (in parentheses) of individuals exhibiting stipple across
all years were: wild grape (32.7%) > Winged Sumac (16.8%) > Chinese
Tallowtree (8.6%), as shown in Table 1. Wild grape and Winged Sumac
were also reported to be useful ozone-sensitive bioindicators in the Edwin
B. Forsythe NWR in New Jersey (Davis and Orendovici 2006). However,
grape foliage in both refuges exhibited late-summer insect injuries that complicated
evaluation of ozone-induced symptoms. Similarly, Winged Sumac
foliage became red during late summer in both refuges, complicating stipple
evaluation, and could not be utilized in the Cape Romain NWR in 1998 and
2003 (Table 1). Photographic images of stipple on wild grape and Winged
Sumac were taken, but quality was not high enough for publication.
Although Chinese Tallowtree was asymptomatic in 1996, this species produced
classic, easily discernable stipple (Fig. 2) in other years, and exhibited
little insect injury or foliar reddening. Anderson et al. (1986) reported that
Chinese Tallowtrees on Bull Island exhibited symptoms resembling those
474 Southeastern Naturalist Vol. 8, No. 3
Figure 2. Classic ozone-induced stipple on the upper surface of a Chinese Tallowtree
leaf from the Cape Romain NWR.
Figure 1. Cumulative sum of hourly ozone concentrations ≥60 ppb (CUM60, ppbhrs)
monitored within the Cape Romain NWR at EPA AIRS Site # 45-019-0046 beginning
April 1 during survey years (1996–1998 and 2002–2003). Monitored ozone
data furnished by David Joseph, National Park Service, Denver, CO.
2009 D.D. Davis 475
caused by ozone, but cautioned, “We do not have any fumigation data on this
species and cannot be sure if the observed symptoms were ozone caused.”
However, based on their symptom description and the author’s observations,
it is likely that symptoms observed by Anderson et al. were caused by ozone.
Chinese Tallowtree warrants consideration for use as an ozone bioindicator
in southern refuges. This assumption should be confirmed in controlled exposure
Ambient ozone concentrations
Annual, ambient, cumulative ozone concentrations (CUM60, ppbhrs)
as of the first day of survey each year were: 24,437 ppb-hrs (1997) >
22,668 ppb-hrs (2002) > 17,769 ppb-hrs (2003) > 17,424 ppb-hrs (1998)
> 9216 ppb-hrs (1996). Within a growing season, ozone concentrations followed
similar patterns each year, gradually increasing from April to the end
of summer and then leveling off in September (Fig. 1). In comparison to
other wildlife refuges surveyed by the author, ambient ozone concentrations
in the Cape Romain NWR were greater than in the remote Seney NWR in
northern Michigan (Davis 2007b), but somewhat less than those at Brigantine
NWR in New Jersey (Davis and Orendovici 2006), Moosehorn NWR in
Table 1. Summary of observations made during the 1996–1998 and 2002–2003 surveys at the
Cape Romain NWR. Numbers within species columns refer to number of plants exhibiting
ozone-induced stipple as compared to the total number of plants evaluated for that species; data
also expressed as percentages. Ozone values are expressed as CUM60 (ppb-hrs) as of the first
day of survey for each year. Drought is expressed as Palmer drought severity index (PDSI) as
of July 31 in each survey year. Blank spaces in species columns indicate no data.
Wild Winged Chinese Weighted ozone PDSI
Survey date/no. plants grape Sumac Tallowtree average (ppb-hrs) (index)
1996 (Aug 21–24)
Number plants examined 56 45 273
Number plants stippled 14 5 0
Percentage 25.0% 11.1% 0.0% 5.08% 9216 -1.17
1997 (Aug 13–18)
Number plants examined 54 73 107
Number plants stippled 24 14 30
Percentage 44.4% 19.2% 28.0% 29.06% 24,437 1.23
1998 (Aug 18–20)
Number plants examined 33 105
Number plants stippled 12 23
Percentage 36.4% 21.9% 25.36% 17,424 -3.05
2002 (Sept 9–11)
Number plants examined 41 25 108
Number plants stippled 13 5 5
Percentage 31.7% 20.0% 4.6% 13.22% 22,668 0.27
2003 (Sept 3–5)
Number plants examined 15 116
Number plants stippled 2 3
Percentage 13.3% 2.6% 3.82% 17,769 2.45
Weighted average 32.66% 16.78% 8.60%
476 Southeastern Naturalist Vol. 8, No. 3
Maine (Davis 2007a), and the Mingo NWR in Missouri (D.D. Davis, unpubl.
data). Ozone data at the latter two NWRs were inferred from the EPA monitoring
site nearest each refuge. Ozone, or its precursors, may be produced in
urban or industrial areas southwest of the refuge and transported to the Cape
Romain NWR on southwesterly winds.
Based on the 1996 observations, the threshold CUM60 ozone level
needed to induce symptoms on sensitive plants within the Cape Romain
refuge is likely near 9000 ppb-hrs. If so, ozone injury might have occurred
on sensitive plants in the refuge as early as June in 2002 and 2003 when
this threshold was exceeded (Fig. 1). Injured leaves from June–July exposures
may defoliate or become hidden by new growth, and may not be
evident during the mid- to late-August period, when ozone-injury surveys
are usually conducted. In addition, high ozone levels in late spring or early
summer may cause injury to plant species that emerge and complete their
life cycles early in the growing season, such as late-spring or early-summer
ephemerals (Davis and Orendovici 2006) or other early successional
species (Barbo et al. 1998); such injury would likely not be evident during
mid- to late-August surveys. The binary logistic model was not used to
examine relationships among species, ozone levels, and drought since the
Pearson’s (chi-square) goodness-of-fit statistic was not significant (Minitab
2003). Lack of significance was likely influenced by the small dataset,
as well as confounding relationships between ambient ozone levels and
In summary, foliar symptoms caused by ambient ozone were observed on
bioindicator plants within the Cape Romain NWR during each survey year.
It is likely that ozone levels are great enough during most years to cause
injury to sensitive plants within the refuge, including those growing within
the Class I Wilderness air quality area. The US Fish and Wildlife Service
may utilize the results of these surveys when making air quality management
decisions, including those related to review of Prevention of Significant Deterioration
permits. Such survey data may be used as input to strengthen our
National Ambient Air Quality Standards for ozone (USEPA 2007).
The author gratefully acknowledges receiving financial support from the US Fish
and Wildlife Service, Air Quality Branch, Denver and the Pennsylvania Department
of Environmental Protection, Bureau of Air Quality, Harrisburg. The author also
thanks Mr. David Joseph, National Park Service, Denver for furnishing the ambient
Anderson, R.L., C. Scarrow, and J.L. Knighten. 1986. Survey for ozone-caused
injury on sensitive plant species on the Francis Marion National Forest and Bull
Island (Cape Romain National Wildlife Refuge). USDA Forest Service, Forest
Pest Management, Asheville Field Office, Asheville, NC. Report 87-1-3.
2009 D.D. Davis 477
Anderson, R.L., C.M. Huber, R.P. Belanger, J. Knighten, T. McCartney, and B. Book.
1989. Recommended survey procedures for assessing ozone injury on bioindicator
plants in Region 8 Class 1 Wilderness areas. USDA Forest Service, Forest
Pest Management, Asheville Field Office, Asheville, NC. Report 89-1-36.
Barbo, D.N., A.H. Chappelka, G.L. Somers, M.S. Miller-Goodman, and K. Stolte.
1998. Diversity of an early successional plant community as infl uenced by ozone.
New Phytologist 138:653–662.
Chappelka, A.H., H.S. Neufeld, A.W. Davidson, G.L. Somers, and J.R. Renfro. 2003.
Ozone injury on Cutleaf Conefl ower (Rudbeckia laciniata) and Crown-beard
(Verbesina occidentalis) in Great Smoky Mountains National Park. Environmental
Davis, D.D. 2007a. Ozone-induced symptoms on vegetation within the Moosehorn
National Wildlife Refuge in Maine. Northeastern Naturalist 14:403–414.
Davis, D.D. 2007b. Ozone injury to plants within the Seney National Wildlife Refuge
in northern Michigan. Northeastern Naturalist 14:415–424.
Davis, D.D., and T. Orendovici. 2006. Incidence of ozone symptoms on vegetation
within a National Wildlife Refuge in New Jersey, USA. Environmental Pollution
Eckert, R., R. Kohut, T. Lee, and K. Stapelfeldt. 1999. Foliar ozone injury on native
vegetation at Acadia National Park: Results from a six-year (1992–1997) field
survey. National Park Service Technical Report NPS/BSO–RNR/NRTR/00–12,
Helm, A.C., N.S. Nicholas, S.M. Zedaker, and S.T. Young. 1991. Maritime forests
on Bull Island, Cape Romain, South Carolina. Bulletin Torrey Botanical Club
Hildebrand, E., J.M. Skelly, and T.S. Fredericksen. 1996. Foliar response of ozone
sensitive hardwood tree species from 1991 to 1993 in the Shenandoah National
Park, VA. Canadian Journal of Forestry Research 26:658–669.
Lefohn, A.S., and W.J. Manning. 1995. Ozone exposures near Class I wilderness
areas in New Hampshire and Vermont. Atmospheric Environment 29:601–606.
Manning, W.J., S.V. Krupa, C.J. Bergweiler, and K.I. Nelson. 1996. Ambient ozone
(O3) in three Class I wilderness areas in northeastern USA: Measurements with
Ogawa passive samplers. Environmental Pollution 91:399–403.
Minitab, Inc. 2003. MINITAB Statistical Software, Release 14 for Windows. State
College, PA, State College, PA, 16801.
Palmer, W.C. 1965. Meteorological Drought. Research Paper Number 45. US Department
of Commerce, Washington, DC.
Richards, B.L., J.T. Middleton, and W.B. Hewitt. 1958. Air pollution with relation to
agronomic crops. V. Oxidant stipple of grape. Agronomy Journal 50:559–561.
Showman, R.E. 1991. A comparison of ozone injury to vegetation during moist and
drought years. Journal of the Air and Waste Management Association 41:63–64.
Skelly, J.M. 2000. Tropospheric ozone and its importance to forests and natural
plant communities of the northeastern United States. Northeastern Naturalist
Stalter, R. 1984. The fl ora of Bull Island, Charleston County, South Carolina. Bartonia
United States Congress (US Congress). 1977. The Clean Air Act as amended August
1977. P.L. 95-95. US Government Printing Office, Washington, DC.
478 Southeastern Naturalist Vol. 8, No. 3
United States Department of the Interior (USDOI). 2003. Ozone-sensitive plant species
on National Park Service and US Fish and Wildlife Service Lands: Results
of a June 24–25, 2003 Workshop, Baltimore, MD. Natural Resource Report NPS/
NRARD/NRR-2003/01. Baltimore, MD.
United States Environmental Protection Agency (US EPA). 2007. Review of the
national ambient air quality standards for ozone. Policy Assessment of Scientific
and Technical Information, EPA-452/R-07-003. Washington, DC.
United States National Climatic Data Center (USNCDC). 2004. Palmer Drought
Severity Index Data for Division 7 of South Carolina. Available online at http://
www7.ncdc.noaa.gov/CDO/cdodivisionalselect.cmd. An older version, which is
no longer available, was accessed 29 April 2004.
Yuska, D.E., J.M. Skelly, J.A. Ferdinand, R.E. Stevenson, J.E. Savage, J.D. Mulik,
and A. Hines. 2003. Use of bioindicators and passive-sampling devices to evaluate
ambient ozone concentrations in north central Pennsylvania. Environmental