AIR HYGIENE REPORT no. 10
Contents

III GASEOUS POLLUTANTS

2 Lichens

  2.1 Introduction
  2.2 Monitoring design

2.2.1 Monitoring method selection
2.2.1.1 Mapping
2.2.1.2 Index of Atmospheric Purity
2.2.1.3 Phytosociological methods
2.2.1.4 Response methods
2.2.1.5 Element bioaccumulation
2.2.1.6 Transplants
2.2.2 Species selection
2.2.3 Sampling methods
2.2.3.1 Lichen cover
2.2.3.2 Lichen frequency
2.2.3.3 Lichen diversity

  2.3 Sulphur dioxide (SO₂)

2.3.1 Species distribution
2.3.1.1 Multi-national surveys
2.3.1.2 National surveys
2.3.1.3 Regional surveys
2.3.1.4 Urban and industrial surveys
2.3.2 Sulphur content
2.3.3 Response methods
2.3.3.1 General lichen health
2.3.3.2 Physiological and biochemical effects
2.3.4 Transplants
2.3.5 Combined methods

  2.4 Fluoro-compounds
  2.5 Nitrogen oxides and ammonia (NO_x and NO₃)
  2.6 Ozone (O₃)

2.1 Introduction

Lichen communities growing on tree bark (corticolous species) and walls and rocks (saxicolous species) show changes in response to air pollutants, particularly sulphur dioxide (SO₂), fluoro-compounds (F), deposition of nitrogen compounds and ozone (O₃). Lichens are particularly useful in indicating pollution loads over long periods (Richardson, 1988).

Literature on lichens and air quality is vast, to the extent that the Lichenologist publishes updated abstracts of the literature periodically (e.g. Henderson, 1994; 1995; 1996a; 1996b). Seaward (1993) examined the history and future of field studies concerned with lichen and SO₂ air pollution. A useful overview of recent developments in the use of lichens as bioindicators up to 1990 was provided by Richardson (1991). Numerous books have been published in this area (Richardson, 1992; Nash and Wirth, 1988).

Will-Wolf (1988) undertook a review of North American air pollution monitoring studies using lichens and/or bryophytes. The review revealed that most studies focused on lichens on trees in eastern deciduous and coniferous forests around a point source of low-moderate level pollution.

Burton (1986) found that the use of lichens in gaseous pollutant monitoring was based mainly on species distribution observations and to a lesser extent on chemical analyses and lichen transplants. Recent developments show an increased emphasis on the use of biochemical and physiological responses as indicators of air pollution, probably due to technological advancement.

This section initially highlights some aspects of monitoring design in gaseous air pollution monitoring using lichens. Few regional monitoring surveys have been undertaken using lichens and surveys are very much centred around urban and industrial areas. References

2.2 Monitoring design

2.2.1 Monitoring method selection

Several gaseous air quality monitoring methods utilising lichens are available. Monitoring can be qualitative or quantitative and employ single indicator species or community changes. The choice of method depends on the purpose of the survey, the size of study area, resources available and the desired detail of the output. Air quality assessments, which observe species distribution patterns, are well recognised. Data on changes in lichen occurrence and abundance at species and community level are subjected to varying degrees of analyses and used to produce maps, identify zones and/or indices of air quality. Other methods reviewed are physiological/biochemical responses as indicators of air pollution, and lichen health has been used as an indicator of air quality degradation. By analysing element content from lichen samples at different distances from a pollution source, the type of pollution and the size of the fallout zone can be determined. Transplantation techniques are frequently used to assess air quality impact in an area.

The basis of these methods is presented in the following paragraphs. Their application and development on multi-national, national, regional and urban scales are illustrated in Sections 2.3 to 2.6.

2.2.1.1 Mapping

Most species distribution investigations involve mapping. Distribution mapping of common and sensitive lichen species is a relatively simple and inexpensive method of air quality monitoring. The method distinguishes areas with varying degrees of pollution. Studies can be in relation to point emission sources such as power plants and smelters, a general source area such as an urban area or industrial complex, or as a means of producing baseline data of a previously unsurveyed site or in pre-development appraisals (Showman, 1988). Species distribution monitoring can investigate spatial or temporal patterns. It is more useful with prior knowledge of lichen sensitivity to air pollutants and historical and/or natural or control data of the study area.

Distribution patterns can include presence or absence of species in response to a pollution gradient, reductions in cover of species and recolonisation of species associated with improved air quality. Distribution studies often include investigations of the health of the lichen flora.

Showman (1988) reviewed North American lichen mapping research up to 1986. This review advised on aspects of study area determination, reconnaissance, site selection, data collection and final distribution mapping. These considerations are paramount in the effective execution of a biological monitoring programme.

A major difficulty in lichen distribution studies in air quality monitoring is the fact that the measured parameters (e.g. abundance/presence/absence/diversity) can be attributed to several factors other than changes in air quality. Other factors include unfavourable habitat conditions, historical reasons, competition pressures, or anthropogenic impacts other than air pollution, such as landscape changes.

A practical progression of species distribution maps was the development of zone mapping. Zone maps classify areas using the number and type of lichen species present, which indicate the extent and/or distance from the pollution source. Burton (1986) reviewed the earliest zone mapping studies. Hawksworth and Roses' (1970) qualitative scale of relating mean winter SO₂ concentrations (µg m^-3) with certain epiphytic lichen assemblages on acidic and basic tree bark remains the key paper on zonal mapping and is cited and utilised extensively in the literature. Ten zones were devised, with zone 1 species indicating SO₂ levels > 170 µg m^-3 and zone 10 representing 'purity'. Although the scale has proved effective in monitoring the spread and extent of SO₂, doubts have been cast on its application in areas encountering reduced levels or qualitative changes in air quality (Richardson, 1988). References

2.2.1.2 Index of Atmospheric Purity

Calculations of Indices of Atmospheric Purity (IAP) examine the effects of a pollutant source on lichen communities. It is a quantitative phytosociological approach requiring the collection of data such as frequency and/or percentage cover and a factor of tolerance to toxicity. IAP values generally increase as communities become more complex further from the pollution source. These values can also be plotted on a map, which in turn can be used to determine IAP zones. Burton (1986) reviewed the use of Indices of Atmospheric Purity in air quality monitoring.

Showman (1988) highlighted the importance of site selection in this type of survey to ensure that lichen patterns are due to air quality differences rather than lichen substrate variations. Will-Wolf (1988), in her review of quantitative approaches to air quality studies, concluded that IAP indices rely on overall linear declines of species in communities affected by air pollutants. Therefore their efficacy as measures of community change are limited when pollutant gradients are shallow, where species dominance shifts are as important as linear declines. On comparison with other phytosociological methods, Wirth (1988) suggested that due to potential error associated with Q (refer to Section 2.3.1.4), regions which are inherently species poor with the same emission impact as lichen rich regions will have lower IAP values. The same author believes that the IAP method is not adequate in differentiating between pollution and climatic impacts.

Modifications of the IAP and developments of new indices are discussed below (Section 2.3.1). References

2.2.1.3 Phytosociological methods

Phytosociological methods in air pollution monitoring study plant communities rather than single indicator species. Air pollution alters community structure, which is reflected by changes in the community composition and coverage. Wirth (1988) reviews the advantages and disadvantages of the phytosociological approaches to monitoring temporal and spatial changes in air quality. The method lends itself better to temporal monitoring than spatial. It is adept at differentiating between climatic/edaphic and air quality impacts on lichen species/community distribution. However, quantitative data collection is inherently more labour intensive, especially over large study areas. The same review presented a phytosociological sensitivity scale (1 to 14) to (acidic) air pollution in southern Germany (Box 3.1), but tolerance values for particular air pollutants were not available. This method is not recommended in obtaining actual pollutant concentrations, but more as an early warning system in environmental protection and in revealing relative changes in air quality over time.

2.2.1.4 Response methods

Additional information in lichen monitoring studies can be gained from observing morphological changes and the health status of lichens. Quantifiable physiological changes can be examined as a measure of pollution stress. The advancement of laboratory techniques has aided the development of such procedures.

2.2.1.5 Element bioaccumulation

Various studies have demonstrated good correlations between non-metallic elemental content in lichens and atmospheric deposition levels of these elements (Burton, 1986). For example, sulphur concentrations in lichen tissue have been correlated to SO₂ contamination in the atmosphere (Garty, 1985). Monitoring elemental accumulations in lichens does not monitor actual effects but can indicate areas of lesser or greater deposition. This method can indicate areas of impact in advance of other detectable effects. References

2.2.1.6 Transplants

Transplantation exercises are frequently used to demonstrate or assess effects of gaseous air pollution on lichens transplanted from a clean to a polluted environment. The transplantation concept has been discussed in Chapter II with regard to heavy metal contamination. Most of the same principles apply to gaseous air pollutant assessment. The use of transplants requires some prior knowledge of the influence of transplantation on the measured response.

2.2.2 Species selection

Different lichen species are chosen for air pollution monitoring depending on their tolerance to air pollutants. The sensitivity of lichens to air quality has facilitated their utilisation as important tools in air quality monitoring. Kovács (1992a) summarised the physiological and morphological features of lichens, which make them more sensitive to air pollutants than higher plants. Features include the absence of a cuticle, low chlorophyll content and lack of excretion.

Awareness of the sensitivity of lichens is important in their function as bioindicators, particularly in mapping studies and transplantations. Richardson (1988) claimed that 'the best way to convince non-biologists of their (lichens) value is by establishing how, why, and at what levels, air pollutants are harmful to lichens'. Sensitivity coefficients are frequently used in the calculation of pollution related indices (Brakenhielm and Qinghong, 1995).

Steubing (1983), cited by Kovács (1992a), summarised the critical SO₂ concentrations for various lichen species published by different authors. In general these authors show comparison with Hawksworth and Roses' (1970) qualitative lichen sensitivity scales (discussed below). These scales of sensitivity have been supported and correlated with the results of controlled laboratory fumigation results (Nash, 1988).

Empirical laboratory and field fumigation exercises to establish actual sensitivity values of lichens to air pollution have often proved unsuccessful (Richardson, 1988). This is due to a combination of maintaining the metabolic activity of lichens in the lab, the need for sophisticated monitoring equipment and simulating low doses over a long period of time. The advent of open top fumigation chambers used for air pollution effects on field-grown plants show promise for lichenologists (Richardson, 1988). These systems overcome many fumigation problems in allowing the control of air quality by adding and subtracting pollutants without creating unacceptable environmental changes. In this way absolute values of lichen tolerance and relative importance of particular gaseous pollutants over longer time periods in field situations can be determined. Bates et al. (1996) used open-air fumigation to investigate lichen colonisation in a newly planted forest subjected to SO₂ and O₃. Small environmental changes may still be observed in open top chambers. These include higher than ambient temperatures, reduced rainfall and unnatural wind speeds (Colls, 1997).

A comprehensive review of the physiological responses of lichens to laboratory air pollutant fumigations is presented in Fields (1988). Various gaseous pollutants were investigated which affected lichen physiological processes in the apparent order of sensitivity: nitrogen fixation > potassium ion efflux/total electrolyte leakage > photosynthesis, respiration > pigment status. This may aid the selection of parameters in monitoring programmes but the application of laboratory results to actual field studies should still be used tentatively.

The sensitivity of a species may vary to an extent on the substrate type. In response to air pollution, species growing on basic substrates will persist longer than species growing on acidic substrates (Hawksworth and Rose, 1976). In the Liphook forest fumigation project, Bates et al. (1996) found that the three lichen species under investigation (Evernia prunastri (L.) Ach., Hypogymnia physodes (L.) Nyl. and Lecanora conizaeoides Nyl.) showed an obvious preference for Norway spruce over Scots pine, the latter containing the most acidic bark. The sensitivity of lichen species may be attributed to other factors such as the tolerance of the contained algal strains, morphology and water relations (Richardson, 1988).

Eight ecological groups of lichens are presented in Kovács' (1992a) review of lichens' role in environmental protection. Attributes of these groups may account for the tolerance of different lichens. Additionally, different reproductive stages of epiphytes show varying pollution sensitivities.

It has been hypothesized that the sensitivities of epiphytic lichens vary between regions. For example, Richardson (1988) suggested that the wetter climates of Sweden and Ireland make species more sensitive to SO₂ than they would be in England. References

2.2.3 Sampling methods

Various sampling methods are available in lichen monitoring, depending on the nature of the method deployed and desired output. Some sampling methodologies used to estimate or measure lichen presence and/or abundance are presented below.

2.2.3.1 Lichen cover

Estimation of lichen cover in relation to pollution patterns is a common procedure and may involve total lichen cover or individual species cover. Some exercises are more time-consuming than others.

Quantification of lichen cover has been undertaken using quadrats and/or photographs. Addison (1984), in a study into the influence of oil sands extraction and processing emissions on lichen cover of transplanted communities, used a 15 x 20 cm gray-card quadrat and photography. In this study lichens were also moistened to minimise errors arising from different lichen hydration states at the time of sampling. The author concluded that when practicing this technique, lichen cover must be high to overcome measurement errors.

Bates et al. (1996) used two methods to estimate percentage thallus cover of the bark in their assessment of lichen colonisation in the Liphook Forest fumigation project, Hampshire, England. For Hypogymnia physodes and Evernia prunastri, thallus area was determined from thalli numbers and diameters, and for Lecanora conizaeoides a scale of one to five corresponding to percentage thallus cover of the bark was used.

Global average cover (GAC) is sometimes estimated as a measure of lichen abundance. This is measured as the average cover of lichen species on all trees sampled (Legrand et al., 1995). References

Sampling of trunk epiphytes was detailed in the manual of integrated monitoring (UN ECE 1993). Three methods have been prescribed for the estimation of epiphyte cover.

• Line method. Lichen cover is read at four levels (60, 90,120 and 150 cm) in relation to trunk circumference. This is achieved by tying a measuring tape (mm) horizontally around the tree with zero to the north and figures increasing clockwise. Overall cover of each species is the mean of individual levels values.
• Point method. A permanent transparent plastic frame (e.g. 30 x 40 cm) divided into uniformly spaced points is placed on either side of the tree trunk. Reported cover is the mean of the percentage of points occupied by each species at each sampling location.
• Visual estimate. Visual estimates of lichen cover can be taken between selected trunk heights.

Correlations between percentage cover of total lichen vegetation and a particular SO₂ level may be erroneous due to the inclusion of toxitolerant species. Selection, observing and/or mapping of either toxiphobous or toxitolerant species associated with a particular SO₂ level would be more successful in terms of pollution monitoring (Seaward, 1993).

2.2.3.2 Lichen frequency

Kovács (1992a) suggested a method for estimating lichen species frequency on a tree trunk. An area of 30 x 130 cm at height approximately 150 cm above ground is chosen. By dividing the area into forty subsections, the number of sections in which the species is observed is determined. A score can be applied to this data.

Lichen frequency on its own is rarely correlated to pollution contamination. This parameter is usually determined for other index calculations such as IAPs. Species frequencies are easily expressed as maps of the study area.

2.2.3.3 Lichen diversity

Some authors have demonstrated correlation of species diversity with pollutant concentrations (Seaward, 1993). In the Netherlands, Van Dobben and De Bakker (1996) obtained stronger correlation between the number of epiphytic lichen species per 5 x 5 km2 grid square and measured SO₂ pollution than with number of species per sample point or species abundance measurements and SO₂ concentration. Species richness is easily obtained from distribution and/or IAP mapping studies.

Other parameters used in lichen distribution and mapping studies in particular include species density (i.e. number of species per unit squared) and luxuriance-density values (Showman, 1988). References

2.3 Sulphur dioxide (SO₂)

2.3.1 Species distribution

2.3.1.1 Multi-national surveys

Epiphytic lichen monitoring is prescribed in the manual: UN ECE International Co-operative Programme on Integrated Monitoring under the Convention on Long-range Transboundary Air Pollution. The onitoring programme was initially in response to increasing awareness of acid rain but now covers other air transported pollutants. Lichen coverage (refer to Section 2.2.3.1) and thalli vitality (Section 2.3.3.1) were proposed as bioindication parameters of 'NO_xious gases and chemistry of precipitation, throughfall, bark and stemflow'. The manual provides prescriptive details on the recommended methodology to be carried out at one to five year intervals (UN ECE, 1993). Some key considerations are summarised below.

• During tree plot selection extreme habitats such as sheltered depressions or wind-exposed heights should be avoided.
• At least one widely distributed species should be utilised within a country or a region for temporal and spatial comparability.
• Select sample trees that are visibly healthy and have stable bark.
• Observations should be made at a trunk height of 50 to 200 cm. All species types within this area should be noted in addition to those receiving a cover value.
• Surveys are supplemented with measurements of certain pendulous lichens, which are a useful gauge of pollutant levels.

The manual defined the following lichen Pollution Sensitivity Index (PSI):

where:
 
D = mean cover per tree
Q = empirical sensitivity factor for each lichen species
i = plot or area
j = lichen species

High index values indicate a high abundance of sensitive species, which implies a low pollution effect.

Standardised site selection criteria, sampling methods, data presentation and quality control measures as prescribed in this manual are key to integrated multi-national air pollution monitoring programmes. This is a reflection of the increasing role of biomonitoring in air pollution monitoring. References

2.3.1.2 National surveys

Sweden is particularly experienced in the use of lichens in air pollution monitoring. Long-term monitoring within the Swedish National Environmental Monitoring Programme (PMK) is undertaken in much the same way as the UN ECE collaborative studies. Lichen cover is estimated by the line method described in Section 2.2.3.1. Permanent marking of sample trees and observation points enable continuous monitoring of the same lichens.

Poor correlations between lichen occurrence and measured pollutants in an area imply that other factors may be responsible for lichen trends. In this way the importance of air pollution in an area in relation to other environmental factors can be assessed. An array of statistical approaches has been implemented to assess such situations. Brakenhielm (1996) and Brakenhielm and Qinghong (1995) reported on PMK results and applied First Principal Components Analysis (PCA) and partial Redundancy Analysis (RDA) to the data. Weighted Mean Sensitivity (WMS) was used as the most effective index of air quality:

where:
 
K_j = sensitivity coefficient for the jth species
n_ij = number of individuals of the jth species along a circle at the ith level
j   = 1,2,… s, the number of species along the circle
N_i = total number of individuals of all species at the i^th level.

Each species was allocated a sensitivity coefficient (on a ten point scale) based on its tolerance to pollutant deposition (Hultengren et al., 1991). For example, the relative tolerance Hypogymnia physodes was assigned a Kj value of 2 whereas the more sensitive Alectoria sannentosa had a Kj value of 7.

Lichens were more strongly influenced by climate and geographic location than pollutant deposition. The different growth rates of Pinus sylvestris between north and south may be responsible. Bark loss due to faster growth rates in the south favours rapid colonisers (e.g. Hypogymnia physodes) over slower colonisers such as Bryoria and Usnea. The authors believed that refinements to Swedish sampling methodology should be made.

Increasingly, species distribution studies are concerned with mapping re-colonisation in areas. This is in response to a decline in SO₂ levels since the 1970s and increases in other pollutants such as NO_x and NH₃. In 1988/89 Van Dobben and De Bakker (1996) updated previous surveys of epiphytic lichens in the Netherlands. Only 17% of the previous area was covered but sampling design was such that a representative picture of lichen trends since the 1970 to 1973 study (De Wit, 1976) was obtained. Sophisticated statistical analyses such as RDA were employed in the recent Dutch study enabling more quantitative relationships between direct air quality measurements to be determined. Two major differences between surveys were apparent:

• Increases in species diversity in the later survey, attributed to decreases in SO₂ levels.
• Increase in the number of nitrophytic species such as Physcia caesia in 1988/89, probably due to bark pH and/or NH₃.

References

2.3.1.3 Regional surveys

The Calibrated Lichen Indication Method is a standard method of evaluating biological effects and total air pollution in the Swiss midlands (Herzig et al., 1989). It evolved as the result of comparisons between twenty variations of the IAP calculation and technical emission data. The model, IAP18 was selected because it produced the highest correlations. The model is based on the frequency of forty selected lichen species. Five zones of effect have been categorised which correspond to five zones of total air pollution (Table 3.3).

Osaka plain in Japan was divided into three areas for the purpose of assessing lichen distribution patterns in relation to air pollution (mainly SO₂ and NO₂) in the region (Hamada et al., 1995). Frequency and cover of five indigenous lichen species were mapped. Lichen frequency was measured as the percentage of trees possessing a specific lichen species of all trees in each grid unit. Lichen cover was categorised according to three classes: > 1/3, > 1/10 to 1/3 and W 1/10. Phaeophyscia limbata displayed relatively high tolerance. Frequencies of this species peaked at intermediate SO₂ concentrations (7 to 8 ppm for this region) and were depressed at low and high pollution areas. In contrast Lecanora pulverulenta proved a better bioindicator, decreasing in frequency with increasing distance from the central, polluted areas. Conclusions were easily drawn with respect to SO₂ pollution. However, this study revealed the comparative lack of knowledge in terms of NO₂ contamination, despite NO₂ levels reaching three times that of SO₂ in the Osaka plain. References

2.3.1.4 Urban and industrial surveys

Despite some regional studies, lichen distribution in relation to urban studies is still the most common application. Additionally, lichens continue to be frequently used to estimate air pollution influences of industrial sources.

Muir and McCune (1988) undertook comparisons of lichens, tree growth and foliar symptoms in detecting air pollution in a polluted area of Illinois and a 'cleaner' area in Indiana. Differences between the two sites were most pronounced in lichen results, which reflected several years of air pollution. Foliar symptoms tended to indicate conditions of the current year. Species richness, total cover and species composition were greater in the least polluted site.

Despite criticism, IAP methods have been practiced extensively in air pollution monitoring, particularly in mapping studies. In Thessaloniki, Greece (Diamantopoulos et al., 1992), IAPs for twenty one sites were calculated according to Leblanc and DeSloovers' (1970) formula:

where:
 
n = number of species present at a site
f = frequency (cover) of the species (scale of 1 to 5)
Q = ecological index of each species i.e. the mean number of other lichen species
growing with the species under study in the surveyed area.

In association with this, a site-by-species matrix was subjected to Detrended Correspondence Analysis (DCA) (Hill and Gauch, 1980) and Polythetic Divisive Technique Two-way Indicator Species Analysis (TWINSPAN) (Hill, 1979). The results of the ordination and classification analyses and IAP calculations were comparable, enabling separation of lichen species into three groups and sites into four zones. Zone A represented the most polluted sites, with Zone D depicting least polluted sites. IAPs ranged from 0 at zone A to 37 at the least polluted site within zone D. Lichen groups 1 to 3 succeeded each other and were characterised by varying degrees of pollution. Zone A showed an absence of epiphytic lichen flora. Zone B was characterised by group 1 lichen species dominated by Physcia adscendens and Xanthoria parietina and lacked group 3 species. The relative frequency of group 1 species is lower and group 3 species are still absent in zone C. In zone D, lichen group 3 species dominated.

Few physico-chemical measurements were available for the study area and conclusions on pollution levels were based on the distribution of lichen zones. A noteworthy observation was the association of the foliose lichen Hypogymnia physodes with only the least polluted sites. In contrast this species has been documented as having moderate tohigh tolerance to SO₂ pollution (Hawksworth and Rose, 1970).

Ammann et al. (1987) discussed a slightly modified form of the IAP, sometimes referred to as the Ammann method. Herzig and Urech (1991) cited by Loppi et al. (1996) illustrated the reliability of this method in monitoring total air pollution and recommended its use in alliance with standard sampling procedures practiced in Italy. Kumer et al. (1991) applied this methodology in a study of air pollution in the Ferrara area (north-east Italy) and Loppi and Corsini (1995) adopted it in a study of air quality in the town of Montecatini Terme, Central Italy. Loppi et al. (1996) applied it in Arezzo in central Italy and Loppi (1996) used it in a study of geothermal air pollution in central Italy. The method was as follows:

• 1 to 4 trees were chosen at each sampling station
• a 30 x 50 cm grid, divided into 10 units of 10 x 15 cm, was placed on the trunk of each tree at a height of 120 to 200 cm, in the part of the bole with the highest lichen density
• all the lichen species were noted together with their frequency (F), i.e. the number of grid units in which the species was present
• IAP was calculated for each grid as the sum of all the frequencies present (IAP = *F)
• Reported IAPs for each station were taken as the maximum value calculated.

Zone maps of air quality were constructed either by a function equation using maximum and minimum IAP values (Kumer et al., 1991) or by the plotting programme SURFER (Loppi and Corsini, 1995). Kumer et al. (1991) detected seven classes corresponding to different levels of air pollution. Loppi and Corsini (1995) and Loppi et al. (1996) detected four zones - Zone A: very high pollution, Zone B: high pollution, Zone C: moderate pollution and Zone D: low pollution. Lichen sensitivities in these studies showed compliance with similar urban studies. Both studies suggested that vehicular traffic was the main source of air pollution in the study areas. This emphasised the transition from SO₂ based pollution to NO_x and CO in towns and cities, and the effectiveness of the Ammann method in detecting other pollutants besides SO₂.

Improvement in air quality in north-west London in 1989 was reflected by recolonisation of some epiphytic lichen species since 1980 (Hawksworth and McManus, 1989). Recolonisation in response to decreased mean winter SO₂levels of 130 µg m^-3 to 29 to 55 µg m^-3 appeared not to follow the normal sensitivity scale sequence (Hawksworth and Rose, 1970). Instead the recolonisation process exhibited 'zone skipping', where species did not return in the sequence that they disappeared. An assemblage of zone 4 to 5 species failed to colonise where more sensitive zone 5 to 7 species had. SO₂ levels had fallen so rapidly over the study period that the conditions necessary for invasion of zone 4 to 5 species did not occur.

A twelve year study of epiphyte recolonisation of Quercus robur in south-east England revealed that recolonisation of oaks was lower than for other tree species such as Salix (Bates et al., 1990). This was attributed to the high bark acidity of older London oaks preventing recolonisation of even the relatively tolerant species. Other trees composed of basic bark possessed higher buffering capacities.

Recolonisation surveys in response to ameliorating SO₂ levels in urban areas are complicated by an array of factors. Recolonisation is affected by the rate of SO₂ decrease, nature and age of the substratum, lichen form (foliose species appear to be more successful in recolonisation than crustose species), competitive nature of lichen, extent of agrochemicals and other pollutants in the area (Seaward, 1993). References

A detailed study of lichen communities in the vicinity of a coal power station at La Robla, Spain, revealed three areas of contamination (Alfonso and Rodriguez, 1994). Little air pollutant data was available but the three areas were characterised below:

• Area I represented the most polluted area. A total of 38 different lichen species were observed. For Quercus pyrenaica, IAP, global average coverage (GAC) and species richness index ranges were calculated at 3.0 to 6.0 x 10^-1, 6.0 to 11.0 and 0.2 to 0.5, respectively. Species development was poorer than in the other areas.
• Area II contained 57 lichen species. This area obtained intermediate indices values in comparison to the other areas.
• Area III was the least polluted area, supporting 64 well-developed lichen species. Highest indices were also observed. IAP values fell in the range 11.9 to 15.8 x 10^-1, GAC ranged from 21.0 to 26.2 and species richness from 0.7 to 1.0.

For future monitoring of the area, the authors recommended a selection of valuable bioindicators for three phorophytes used in the study.

2.3.2 Sulphur content

Lichen sulphur (S) concentrations are often measured in association with trace element accumulation studies.

Bruteig (1993) studied atmospheric sulphur and nitrogen deposition in Norway as part of an ongoing programme established to monitor deposition and effects of long-range transported pollutants on various ecosystems and organisms. The author highlighted the importance of sampling strategy and presented detailed procedures for sample collection and analysis, which took place in 1990. Sulphur content in Hypogymnia physodes ranged from 0.046 to 0.183% sulphur of lichen dry weight. Regression analysis demonstrated that sulphate deposition accounted for most of the variation in measured sulphur concentration.

Burton (1986) concluded that sulphur isotopic ratios provided a useful method of assessing main sources of S in lichens. Takala et al. (1991) undertook a large-scale sulphur isotope study of epiphytic and terricolous lichens in non-polluted areas in Finland. The lichen species Hypogymnia physodes showed highly significant correlation between S isotope composition and S content.

A regional gradient in lichen S content was observed by an intensive study in eastern Canada (Zakshek et al., 1986). Highest concentrations (959 µg g^-1) were observed near a smelting complex, and decreased progressively heading east to 329 µg g^-1 within the remoter areas of the region. Thalli S content displayed agreement with sulphate deposition measurements in the study area. Lichen S concentrations in eastern Canada in comparison to north-western Canada reflected the higher pollution burden in the east. References

2.3.3 Response methods

2.3.3.1 General lichen health

Reduction in fertility, injured thalli and absence of young thalli are indicative of air pollution degradation (Wetmore, 1988). Other external changes listed by Kovács (1992a) included change in thallus colour, reduction in thallus size and changes in the thickness of the thallus.

The contained algal part of lichens is responsible for lichen sensitivity between species (Richardson, 1988). Increases in the number of dead and plasmolyzed algal cells and decreases in size and number of regenerative algal cells have been used to measure lichen injury in response to air pollution (Kovács, 1992a).

The same author also presented a table where the rate of degeneration of lichen thalli due to SO₂ pollution was correlated to damage to higher plants. For example, a rate of lichen degeneration of 10 to 35% represented chlorosis and necrosis of the leaves of conifers and cultivated plants. Increasing rates of degeneration implied greater impacts on agriculture, sylviculture and horticulture.

Many European countries utilise vitality scales in assessing the health of lichen thalli (UN ECE, 1993). Five vitality classes are expressed:

1. normal
2. slight damage
3. distinct damage
4. severe damage
5. dead.

Caution should be exerted in lichen health studies to ensure that damage is the result of air pollution and not of other environmental factors.

2.3.3.2 Physiological and biochemical effects

Some comments on physiological and biochemical effects of air pollutants on lichens were mentioned in Section 2.2.2. Physiological and biochemical responses of lichens can be assessed by laboratory and field studies. Field studies under natural conditions are necessary in determining long-term effects of air pollution on lichen metabolism. The basis of laboratory experiments is to confirm under controlled conditions the implied effects observed in the field. Brown (1995) details the interpretive problems associated with cryptogam physiology when measuring characters such as gas exchange, chlorophyll stability and free radical protective systems in the laboratory. Fields (1988) provided a comprehensive overview of the physiological responses of lichens to air pollutant fumigations. Brown (1995) details the interpretive problems associated with field fumigation studies. A whole spectrum of literature is available with respect to air pollutant damage to lichens. This includes Richardson (1988, 1991), Kovács (1992a), Seaward (1993) and Calatayud et al. (1996). The following paragraphs will concentrate on effects on lichens, which are applicable to biomonitoring in the field.

In Rijeka City, Croatia, the extent of cell membrane damage in lichens was correlated with established lichen pollution zones (Alebic-Juretic and Arko-Pijevac, 1989). The laboratory procedures are detailed in the paper and entailed measuring electrolyte leakage (mainly K+ ions) from the prepared lichen specimens, and specific conductivity in leachate when immersed in deionised water. Parmelia tiliacea collected from sites in the lichen desert and struggle zones showed the highest specific conductivity and K content in the leachate. Comparison of lichen species at a coastal control site, 25 km away from Rijeka, showed that the most sensitive species, Parmelia perlata, exhibited the most membrane damage whereas the most resistant species, P. Saxatilis, was regarded as healthy. Results were tentatively correlated with SO₂ concentrations. Humidity plays a role in lichen damage measured as electrolyte leakage, which is significant in semi-arid and arid locations (Rope and Pearson, 1990).

A similar investigation was undertaken within the city of Biel, Switzerland (von Arb and Brunold, 1990). Various physiological responses were measured in naturally growing lichens collected from sites within five defined pollution zones. Annual growth rate of Parmelia sulcata was seven times higher and statistically greater in the low pollution zone than in the critical total air pollution zone. Transfer of C-assimilates from algal to fungal part of the lichen decreased significantly (fifteen times less) between the urban and suburban zones. Chlorophyll-a content in lichens increased significantly within the higher pollution zones in the city and was attributed to elevated NO_x emission from vehicles. Marked changes in the rate of net photosynthesis and dark respiration were not as evident between varying pollution levels. References

Silberstein et al. (1996a) used a variety of physiological parameters to distinguish relative sensitivities of two lichen species (Xanthoria parietina and Ramalina duriaei) in Israel. By transplanting R. duriae from clean locations to polluted areas where X. parietina persists, comparisons between the species were made. The possible protective mechanisms of Xanthoria parietina are discussed in Silberstein et al. (1996b). The different physiological responses observed between the two species may provide further use of lichens as bioindicators. These are listed below:

• Red autofluorescence in photobiont cells indicate healthy cells. Changes in colour to brown, orange and then white under the UV microscope indicates increased pollution damage to the algal cells. X. parietina in the polluted and clean environments exhibited red autofluorescence, whereas only R. duriae from the clean site demonstrated red autofluorescence.
• Chlorophyll degradation was represented by the ratio of optical density at 435 and 415 nm (OD 435/415) in pigment extracted with dimethyl sulphoxide (Ronen and Galun, 1984). R. duriae showed a reduction in OD 435/415 ratio from the clean to polluted site. This parameter has been suggested as more appropriate than chlorophyll-a content (Kardish et al., 1987).
• A significant decrease in photosynthetic rate (measured by photo-acoustic spectrophotometry, Ronen et al., 1985) was observed in transplanted R. duriae. This was not consistent with results obtained by von Arb and Brunold (1990).
• Electrolyte and potassium leakage from lichen membranes can also be used as an indicator of SO₂ contamination as mentioned previously (Alebic-Juretic and Arko-Pijevac, 1989). Silberstein et al. (1996a) found a significant increase in electrolyte leakage in R. duriae from the clean to polluted location.
• ATP content (Silberstein et al. 1990) decreased significantly only in transplanted R. duriae. Kardish et al. (1987) discovered similar trends when R. duriae was transplanted from a clean site to a selection of monitoring sites. ATP content in lichen thalli decreased with increasing pollution at one site. In the same study ATP content proved a much more sensitive indicator of pollution damage than chlorophyll degradation, despite the more time-consuming procedures involved.

The benefit of the latter two parameters is that they are related to the whole lichen and not just the photobiont. The other parameters are indicative of changes in the algal part only (Silberstein et al., 1996a).

The production of stress-ethylene by lichens is another potential physiological response which could be used in bioindication of air pollution in communities in their natural habitats or as a measure of damage in lichen transplants.

In conclusion, alterations to biochemical and physiological mechanisms are useful early detectors of air pollution. However, natural conditions may also affect many internal processes and the inclusion of healthy specimens within the monitoring programme is always advisable. References

2.3.4 Transplants

The following section addresses recent lichen transplantation studies only in the context of bioindication and/or biomonitoring of air pollution.

In the city of Cordoba, Argentina, the lichen Ramalina ecklonii was transplanted to 24 different urban sites along three transects of varying traffic density (Levin and Pignata, 1995). Lichen samples collected from a 'clean' site north-west of the city were hung in nylon bags at a height of three m for eight weeks, prior to analysis. Chlorophyll, phaeophytin, conjugated dienes concentration, soluble protein content and thalli sulphur content were used as bioindication of air pollution in the study area. Assessment of the effectiveness of each parameter would have been useful. Although differences between transects were observed, no indication of decreasing effects from distance from pollution sources or pollution gradient was apparent. A potentially useful Pollution Index (PI) for each transplant was employed in the study defined as follows:

where:
 
Pa = phaeophytin-a concentration (mg g^-1 dry mass)
Ca = chlorophyll-a content (mg g^-1 dry mass)
S_t = sulphur content of transplant (mg g^-1 dry mass)
S_c = sulphur content of control lichen (mg g^-1 dry mass)
CD_t = conjugated diene in transplant (mmol g^-1 dry mass)
CD_c = conjugated diene in control (mmol g^-1 dry mass)

On the basis of the above results, a later more intensive study focusing on one sector of the city of Cordoba, Argentina was undertaken by Gonzalez et al. (1996). The investigation obtained more detail of the traffic and industry related pollution in the area. Sulphur content in lichens was considered a good indicator of heavy traffic sites and PIs proved effective in distinguishing between sites with heavy industrial activity. Vehicular traffic was regarded as the most significant source of pollution in the study area.

A recent development in lichen transplant methodology was 'the culturing of epiphytic lichens on inert non-absorbent materials' which enabled 'easy and repetitive determination of biomass growth rates' (McCune et al., 1996). These authors proposed effective modifications of this technique and discussed biomass growth rate calculations. The developments play an important role for air pollution monitoring using transplants, enabling other parameters such as biomass growth rate to be compared between clean and polluted areas.

2.3.5 Combined methods

Wetmore (1988) proposed a combination of lichen methods for the rapid assessment of air quality in large areas, such as national parks in the United States. The so called 'floristic method' involved collection of samples of all lichen species at each chosen site for identification and elemental analysis, and notation of the health of the lichens at each locality. Lichen health was assessed by observation of symptoms associated with air pollution damage: dead or injured thalli, abnormal growth patterns and frequency of fertile thalli. Details of lichen habitat such as substrate and habitat disturbance were also included in the fieldwork programme. Lichen data was compared with historical records of the area, with flora of the same region in a known area of clean air and the natural geographical distribution and habitat requirements. This established absent species which would normally occur in the study area. Wetmore (1988) postulated that if these species were regarded by the literature as sensitive to air pollution, this could be a possible explanation for the lack of such species. A distribution map of the most sensitive species could then be produced and compared with the distribution of thalli with elevated element concentrations. Wetmore (1988) concluded that the floristic method could provide useful baseline data for the whole lichen flora in a large area, particularly in the United States where lichen flora data is limited in comparison to Europe. The author also stressed its benefit as a screening tool to determine areas requiring further investigation. In these respects the floristic method would be useful in the creation of databases for long-term monitoring of air quality changes, the application of which is growing in importance for internationally and nationally protected areas. References

2.4 Fluoro-compounds

Available data was limited with regard to the assessment of fluoro-compound contamination utilising lichens.

Most studies are concerned with accumulation of fluoro-compounds in relation to a point emission source. However, distinct visible injury of lichens in response to hydrogen fluoride (HF) has been observed (Kovács, 1992a). Lichens turn grayish-white in colour, colony size is reduced and finally colonies separate.

Rope and Pearson (1990) reported levels of fluorine in Lecanora melanophthalma two to four times greater at the Idaho National Engineering Laboratory sampling sites than at two reference sites approximately 60 km away.

In Anglesey in Wales, monitoring of lichens in 58 permanent quadrats in the vicinity of an Aluminium works had been conducted since the 1970s (Perkins, 1992). Response of saxicolous lichens was slower than observed in corticolous species. Fluoride concentration in thalli reached 396 µg g^-1 at well-exposed sites 0.6 km from the works. Percentage cover and fluoride content of Ramalina sp. varied depending on exposure of sites and distance from works. Percentage cover and fluoride content in lichen thalli were closely correlated. Using regression analysis it was predicted those fluoride concentrations of 300 µg g^-1, 100 µg g^-1 and 50 µg g^-1 would correspond to 46, 15, and 10% loss in lichen cover per year. This work is an example of a coherent long-term data set enabling the most appropriate bioindicator species to be sought, exposure of long-term trends and prediction models to be determined.

2.5 Nitrogen oxides and ammonia (NO_x and NH₃)

Nitrogen deposition is increasing in many areas primarily due to increased road traffic intesity and further laboratory and field fumigation experiments and reasearch are needed to confirm the effects of NO₂ on lichens.

Symptoms in response to NO₂ exposure include the production of dark bodies in the vacuoles of algal and fungal parts of lichens (Richardson, 1988). Field fumigations in Sweden found that nitrogen fixation in the nitrogen fixing lichen Peltigera aphthosa increased when exposed to nitrogen in neutral solution, and a combination of ammonium and sulphuric acid had deleterious effects on the lichen (Hallingback and Kellner, 1992).

In the Norwegian study of nitrogen and sulphur deposition in the epiphytic lichen Hypogymnia physodes mentioned previously, Bruteig (1993) discovered nitrogen content levels in the lichen thalli in the range 0.42 to 1.96% lichen dry weight. This value was ten times that of thalli sulphur content. N/S ratios were higher in southern Norway and correlated well with the estimated N/S deposition ratio in Norway. Regression and correlation analyses indicated that most of the variation in lichen N content was attributed to annual mean nitrate concentration, annual mean concentration of ammonium in precipitation and annual wet deposition of ammonia. The aim of the survey was to provide a large-scale spatial picture, and results showed that long-range transported nitrogen from central Europe and Britain contributed significantly to the nitrogen content in Hypogymnia physodes. In addition, this study emphasises the growing importance of nitrogen compounds in air pollution over the past fifteen years. References

2.6 Ozone (O₃)

There was also paucity in the literature in relation to lichen bioindication and O₃. Some fumigation exercises have been reported.

The effects of CO₂ and O₃ on green-algal lichens (Parmelia sulcata) under controlled laboratory conditions were investigated by Balaguer et al. (1996).

Fumigation of two lichen species with SO₂, O₃ and a combination of both, suggested that O₃ was more phytotoxic than SO₂. The combination of gases produced different ultrastructural changes than exposure to either pollutant alone (Eversman and Sigal, 1987).

In the Liphook forest fumigation project mentioned previously, effects of O₃ on lichen colonisation were also investigated (Bates et al., 1996). No marked changes in the abundance of colonising lichens in response to O₃ fumigations were observed. References

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