II Heavy Metals 4

Higher plants have appeal as indicators in air pollution monitoring in highly polluted areas where lichens and mosses are often absent. Higher plants act as biomonitors in the assessment of aerial heavy metal contamination by means of their bioaccumulative properties. Therefore, mainly analytical approaches are used in monitoring of metals.

Metal aerosols pollute soil and plants. Higher plants not only intercept pollutants from atmospheric deposition but also accumulate aerial metals from the soil. Aerial heavy metal deposit are taken up from the soil by plants via their root system and translocated to other regions of the plant.

Particle deposition on leaf surfaces may be affected by a variety of factors, including particle size and mass, wind velocity, leaf orientation, size, moisture level and surface characteristics (Bache et al., 1991). The deposited particles may be washed by rain into the soil, resuspended or retained on plant foliage. The degree of retention is influenced by weather conditions, nature of pollutant, plant surface characteristics and particle size (Harrison and Chirgawi, 1989). Harrison and Chirgawi (1989) demonstrated experimentally the significance of foliar accumulation and translocation of air derived metal pollutants. The foliar route was found to be of similar importance to the soil-root pathway. References

Heavy metal absorption is governed by soil characteristics such as pH and organic matter content (Csintalan and Tuba 1992; Jones 1991). Thus, high levels of heavy metals in the soil do not always indicate similar high concentrations in plants. The extent of accumulation and toxic level will depend on the plant and heavy metal species under observation. In an investigation of Cd, Cu, Ni and Pb uptake from air and soil by Achillea millefolium (milfoil) and Hordeum vulgare (barley) in Denmark, Pilegaard and Johnsen (1984) concluded that Cu and Pb plant concentrations correlated with aerial deposition but not with soil concentrations. In contrast, Ni and Cd content in the plants correlated with deposition and soil content. The distribution patterns and budgets of heavy metals within forest trees growing at contaminated sites in Germany were investigated by Truby (1995).

The interpretation of analytical data is therefore complicated by many factors. However, metal accumulation in plants can reflect the relative extent of the burden and its dispersal.

Plants also demonstrate morphological and physiological responses to heavy metal pollution, some of which may be utilised in bioindication. References

4.2 Monitoring design

For convenience, species selection can be separated into two groups: herbs/grasses and trees/shrubs. Sensitive species are more appropriate where the measurement of plant effects and responses are to be used as bioindication of air pollutants. Accumulative bioindicators tend to be more tolerant to metal loading.

Kovács (1992b) recommended the use of ruderal plants as bioaccumulative indicators due to their ability to accumulate metals in high quantities without visible injury. Ruderals are also widespread plants enabling comparison between regions. Pilegaard and Johnsen (1984) chose to study metal uptake by milfoil (Achillea millefoilium) because of its large surface area.

Some plant species may be more efficient in retaining atmospheric metal particles than others. A measure of this efficiency can be resolved by calculating air accumulation factors (AAF) according to the following equation:

AAF (m³g^-1) = PAc (mg g^-1 dry weight)/CA (mg m^-3)
Where: PAc = atmospheric contribution of the metal in plants
CA = concentration of the metal in the atmosphere.

In their study of tropical plants growing in an industrial area of India, Rao and Dubey (1992) discovered that the degree of accumulation differed substantially between the five species under study. Bache et al. (1991), in their study of metal concentrations in grasses in relation to a municipal refuse incinerator, suggested that the nature and area of the leaf surface would affect foliar deposition. The same authors recommended that this is grounds for the use of consistent sampling of the same plant species at similar times of year and same life cycle stage during plant biomonitoring surveys. References

Both coniferous and deciduous trees can be used in the detection of aerial heavy metal pollution. Coniferous trees indicate pollution over a longer time period. Growth rings may reflect annual variations in metal concentrations in the surrounding environment.

Broad-leaved tree species regarded as sensitive to metal contamination include Betula pendula, Fraxinus excelsior, Sorbus aucuparia, Tilia cordata and Malus domestica (Kovács, 1992c). Numerous bioaccumulative indicators exist. Some examples include Ailanthus glandulosa, Celtis occidentalis, Salix alba, Tilia tomentosa, Sambucus nigra, Quercus robur and Fagus silvatica (Kovács, 1992c). Populus nigra sp. Italica (Italian poplar) has been recommended as a particularly suitable bioindicator of heavy metal burden in Europe (Kovács, 1992c). Amongst many of its appropriate features, this species is genetically homogeneous, easily identifiable and ubiquitously distributed. Robinia pseudoacacia (black locust tree) was recommended as a suitable bioindicator of heavy metal contamination in Hungary (Kovács, 1992c).

The leaves of Rosa rugosa have been reported as effective detectors of rare elements (Kovács, 1992b). References

Coniferous trees are often regarded as better temporal bioindicators of environmental contamination, as their wood type reduces the lateral transfer of contaminants between rings (Zayed et al., 1991).

As with herbs and grasses, tree leaf surfaces may govern the extent of accumulation of particles. In Greece, Sawidis et al. (1995) studied a selection of tree species as biomonitors of Zn and Cu. The investigators discovered that the strongest metal accumulators possessed rougher surfaced leaves which gave rise to the effective trapping and retention of particles.

The size of the area under study will generally determine the spatial distribution of sampling sites. The depth of the study will determine the density of sampling locations. Site selection will also depend on the type of sampler. Site selection criteria should be consistent throughout a survey.

In the Netherlands tree-bark samples were collected from locations arranged along six straight line transects across the country (Kuik and Wolterbeek, 1994). This can be achieved relatively easily for tree sampling because tree/forest locations are readily identified.

In larger scale surveys, for example on the national scale, location of sites near point sources of air pollution is avoided. For example, in a national study in Poland, Dmuchowski and Bytnerowicz (1995) chose sampling sites at least two km away from direct emission sources and at least 300 m from highways.

For comparative studies it is important that sampling is undertaken at the same time of the year to reduce variability. Chemical composition of foliage varies with season and rainfall (Taylor et al., 1990). This is important when sampling annuals and deciduous trees.

Standard sampling of heavy metal accumulation in Populus nigra in central Europe is carried out in August. For most deciduous species this is the time of year when metal content in leaves will be highest. Sawidis et al. (1995) found higher mean Cu and Zn concentrations in autumn compared to spring in the foliage of a variety of tree species. Only the evergreen species, Ligustrum japonicum, which possesses a two year leaf ageing process, showed insignificant seasonal differences in Cu and Zn content.

Throughout a monitoring programme, sample collection should be standardised. The same plant parts from consistent plant heights from the same species of similar height should be utilised.

Metal content will vary depending on which part of the plant is sampled. For example, in herbaceous plants, roots and leaves retain higher metal concentrations than stems and fruits (Kovács, 1992b; Csintalan and Tuba, 1992). The extent of accumulation in different plant parts will vary with species and the nature of the element. Chemical composition varies not only with the age of the plant itself but also with the age of the leaf/needle. Second year needles of balsam and spruce contained significantly higher Hg levels than first year needles during an intensive sampling study in Ontario, Canada (Rasmussen et al., 1991). References

In trees, metal concentrations in needles/leaves have been recorded which are three times that in twig tissue of the same branch (Rasmussen et al., 1991).

Tree bark is appropriate in indicating longer term air pollution. Bark is exposed to air pollutants either directly from the atmosphere or from stemflow. The changes in the chemical composition of the surface layers can be documented. Kuik and Wolterbeek (1994) proposed the use of tree bark samples as biomonitors of heavy metal pollution in the Netherlands. Their use was recommended for larger scale surveys because of their greater availability compared to lichens and mosses. The collection of a large number of samples is more beneficial for the analysis of data by factor analysis (Kuik and Wolterbeek, 1994). The same authors suggested that field sampling procedures of tree bark were simpler and less time-consuming than those practised for lichens and mosses. Bark flakes of about five mm thickness at 1.5 m above ground level were cut. Bark sampling also does not damage the tree (Poikolainen, 1997).

Zayed et al. (1991) used an incremental corer to remove xylem samples from black spruce for Al analysis. By dividing wood samples into two-year sections possible temporal changes may be determined. However, in a study of distribution patterns of heavy metals in forest trees on contaminated sites in Germany, Truby (1995) found no relationship between the radial distribution in the tree rings and the historical heavy metal deposition in the area. The author therefore recommended that xylem rings should not be utilised in the determination of air pollution history at forest sites.

Kovács (1992c) reported on standard sampling methodology for Populus nigra accumulation studies in central Europe. Eight branches are removed at height 5.5 m of solitary unshaded trees. Three one-year shoots are cut from each branch and one leaf is removed from each of these. This results in a total of 24 leaves for analysis of heavy metal content. Similar standard procedures are conducted on Robina pseudoacacia in Hungary.

Lin et al. (1995) used twenty one-year old shoots from branches about five to six m above ground for analysis of metal concentration in balsam fir foliage in Quebec. References

Soil often contains higher metal concentrations than exposed parts of herbaceous plants and thus root analysis is frequently recommended during contamination assessment exercises (Kovács, 1992).

Replication is vital since pollutant concentration can vary even within species (Taylor et al., 1990).

An assessment of within-site variation in metal content in the study has been recommended by some authors (Rasmussen et al., 1991; Lin et al., 1995). In southern Quebec, Lin et al. (1995) tested variability in the elemental concentration in balsam fir needles between individual trees at the same site. Coefficients of variation of < 50% for all elements were regarded as acceptable.

Great variation exists between sample preparation in terms of washing procedures. Priority attention to this issue has been given to leaf samples in particular. Analysis of washed leave samples provides elemental concentration in leaf tissue. Alternatively, elemental content of unwashed leaves will reflect leaf surface and leaf tissue content.

Analytical procedures are similar to those applied to moss and lichen samples, e.g. atomic absorption spectrometry (AAS), neutron activation analysis (NAA) and inductively coupled plasma mass spectrophotometers (ICP-MS). Standard quality control procedures are regularly applied to these laboratory techniques. For example Lin et al. (1995) used standard reference material, NBS-SRM 1575 (pine needle) during NAA measurements of metal burden in forests. References

4.3 National surveys

The following paragraphs discuss three national surveys constituting very different sampler types and approaches. Studies are concerned with sources and patterns of pollution rather than actual metal concentrations.

In the Netherlands, analysis of metal content in tree-bark samples indicated trends in heavy metal concentrations in the country (Kuik and Wolterbeek, 1994). Application of factor analysis supported evidence regarding pollution sources highlighted in previous lichen surveys (Sloof and Wolterbeek, 1991). A criticism of bark sampling in aerial heavy metal deposition monitoring is in the lack of distinction between contributions from soil and airborne sources. In the Netherlands study, factor analysis established metal contribution from soil. Generally, elemental concentrations in bark were lower than those observed in lichens in the 1986/87 survey. For example, Ni mean concentration in bark was 11.0 ppm, which represented a bark/lichen ratio of 0.68. However, some elements displayed higher concentration in bark than lichen samples. For example, Cd showed a mean bark content of 3.1 ppm and a bark/lichen ratio of 1.07. Until fairly recently, the use of tree bark in biomonitoring had been restricted to smaller scale urban and industrial areas. This Dutch study demonstrated the potential of this biomonitor on a larger scale.

A national survey of trace-metal content in the leaves and roots of Taraxacum officinale (dandelion) in Poland demonstrated the value of this plant as a bioindicator of airborne contamination (Kabata-Pendias and Dudka, 1991). Performance of Analysis of Variance, Tukey's test of significance and multiple regression on the data revealed distribution patterns in dandelion metal content throughout the country. In general, trace-metal concentrations in leaves and roots were higher in the industrialised south-western part of Poland than in the rural north-east. Calculation of the ratios of metal concentration in leaves and roots in the whole country, the south-west and north-east provided an indication of which metals originated from airborne pollution. All ratios where greater than 1 and statistically significantlly higher element concentrations were observed in dandelion leaves compared to root samples. Ratios increased from north-east to south-west for Cd, Pb and Zn, implying a significant aerial input of these metals as opposed to root uptake from soil and translocation to leaves. References

A different approach was used in a later Polish survey where analysis of Scots pine needles were used to characterise metal contamination in the country as a whole and in the city of Warsaw (Dmuchowski and Bytnerowicz, 1995). Three zones of Cu pollution were mapped throughout the country, four zones were apparent for Pb, five geographical zones of pollution were established for Zn and Cd and six zones represented As pollution. Zones are summarised in Table 2.9. This survey did not utilise statistical techniques but the use of digital mapping and database production proved an effective alternative in evaluating possible threats to humans and the environment.

Zone	Zn		Cd		Pb		Cu		As
	ppm	%	ppm	%	ppm	%	ppm	%	ppm	%
I	<70	62.0	<0.5	91.2	<10	94.1	<5	99	<0.3	50.7
II	71-100	29.6	0.5-1.0	4.8	10-20	5.1	5.1-10		0.3-1.0	42.8
III	101-130	4.2	1.01-2.5	2.9	20-30		>10	1	1.1-2.0	4.5
IV	130-250	3.6	2.51-5.0	1.1	>30	0.2			2.1-3.0	1.5
V	>250	0.6	>5	0.3					3.1-4	0.45
VI									>4.1	0.1

An array of vegetative types were used to establish regional and temporal changes in atmospheric deposition patterns of Zn, Cu, Pb and Cd in Norway over the decade 1982 to 1992 (Berthelsen, 1995). The study concentrated on forest and ombrotrophic bog sites located in southern and central Norway. Temporal and spatial variations were determined by the calculation of ratios between metal concentrations in 1992 and 1982, supplemented by t-test analysis. All element levels in plants were elevated in southern Norway in comparison to the central region of the country. References

Ratios of approximately 1 and the lack of significant t-tests suggested little change in Zn, Cd and Cu levels in plants over the study period. However, this did not reflect the decrease in deposition of these metals in southern Norway between 1982 and 1992. This was explained by enhanced root uptake of these metals from long-term contaminated soils subjected to heavier air pollution in the south. In contrast, Pb levels in plants strongly reflected both decreased atmospheric Pb deposition from 1982 to 1992 and increased deposition in southern Norway. Therefore changes in Pb concentration were reflected much more quickly than changes in concentrations of other atmospheric contaminants.

This study demonstrated the aptness of even the simplest statistical analyses in drawing conclusions on a national scale. Only the exposed plant parts were analysed in the study and it might have proved advantageous to also analyse roots to support inferences regarding the role of roots in element uptake. References

4.4 Regional surveys

Most regional surveys are associated with forest ecosystems over large areas. Tree bark and foliage are commonly used as bioindicators of heavy metal contamination in forest studies.

Poikolainen (1997) used bark samples from Scots pine collected along seven transects in northern Finland (Lapland) to illustrate heavy metal distribution trends in the area. Heavy metal concentrations were reasonably low throughout the region, and high Cu, Ni and Cr concentrations were only associated with emissions from the Kola Peninsula and industry in south-west Lapland.

Huhn et al. (1995) analysed bark samples from 60-year-old Scots pines to evaluate heavy metal deposition in forest ecosystems in central Germany in comparison to background forest sites. Correlations and factor analyses revealed four metal groups. Fe, Ni, Cr, Cu and Pb were emitted as industrial dust and fly ash and accumulated well in bark. Zn and Cd were associated together and Mn and Hg formed the other two groups. The authors proposed the methodology as a diagnostic tool in monitoring air pollution damage to forests.

Metal analysis of balsam fir foliage demonstrated that trace-metal damage to forests in southern Quebec was not severe (Lin et al., 1995). Trace-element content varied with forest location and elevation. References

4.5 Urban surveys

By analysing Pb and Cd levels in rose-bay leaves (Nerium oleander), Hernandez et al. (1987) characterised the city of Madrid, Spain into four different areas of contamination. Levels of Pb ranged from 13.63 ppm in the lowest contamination zone to 74.25 ppm in the very high contamination zone.

A selection of tree species were sampled from 12 sites throughout Thessaloniki city, Greece, to assess their value as biomonitors of heavy metal pollution (Sawidis et al., 1995). Trees nearer the city centre contained higher metal levels than trees 15 km away. Cu showed a narrow mean concentration range from 5 to 10 mg kg^-1 and showed little variation between tree species. Pb content ranged from < 1.5 to 4.5 mg kg^-1and was highest in Populus alba (white poplar) and Populus niger (black poplar). Poplar leaves also contained the highest Zn concentrations. Overall Zn displayed mean concentrations from 19 to 85 mg kg^-1.

Gradients of pollution of Cu, Pb and Fe were demonstrated in Naples, Italy, by analysing metal content in leaf surfaces and tissue of Quercus ilex (Holly oak) (Alfani et al., 1996). Metal concentrations were significantly higher in leaves from roadside sites than in leaves collected from town squares, which in turn were significantly higher than concentrations measured in urban park trees. Positive correlations between Pb, Cu and Fe concentrations in leaf tissue and leaf surface demonstrated the significance of deposition to leaf tissue content. This supports the theory that aerial deposition to leaves is an important source of metal contamination in leaves. Further evidence was the lack of correlation between metal content in leaves and in soil. Soil samples possessed a higher metal burden than leaf samples. The authors postulated that soil was an appropriate indicator of long-term metal deposition but its utility is limited in the assessment of metals which are highly mobile or major components of soil. References

The epiphytic monocot of the genus Tillandsia has been used as an effective biomonitor of trace-metal pollution in Latin America. Tillandsia species are common throughout Latin America and are similar to lichens and mosses in that their roots do not play an absorptive role. Instead, Tillandsia absorbs water and nutrients directly from the air by means of trichomes present on the leaf surface. Tillandsia caput-medusae was analysed for Cu, Pb and Cd bi-monthly in San José city, Costa Rica to provide an overview of the atmospheric heavy metal burden in the city (Brighigna et al., 1997). Metal concentrations were higher in the urban sampling area and external leaves were more exposed to air than internal ones. Highest metal concentrations corresponded to the dry season. The extremely high Pb content in leaves in comparison to the other two metals confirmed that vehicular traffic was the most important source of air pollution within the city. References

4.6 Line point surveys

Pb accumulation in leaves is a direct reflection of its deposition level. It is a non-essential plant element and deposition and accumulation on plant leaves is its primary route of uptake. Therefore analysis is not complicated by the root uptake and translocation processes apparent for other metals. Furthermore, analysis for Pb is made easier by the fact that concentrations are often unaffected by washing procedures. For example, Hernandez et al. (1987) discovered that the amount of Pb measured in samples of rose-bay leaves showed no difference when the samples were washed or unwashed prior to analyses.

In Madrid, Spain, Hernandez et al. (1987) found significant positive correlation between lead levels in rose-bay leaves and traffic density at sites. However, Sawidis et al. (1995) in Thessaloniki reported that Pb levels were not proportional to traffic density, and higher levels of Pb were associated with sites near road junctions.

Other specific studies have been undertaken directly in relation to highways and their effects on plants. Albasel and Cottenie (1985) found that Cu, Zn and Pb concentrations in plants decreased with increasing distance from major highways in Belgium. Concentrations of Pb were particularly pronounced in comparison to control plants in rural areas. In Finland, Ylaranta (1995) found that the lead concentration of wheat, Italian rye grass and lettuce was 1.5 to 3 times higher 22 m from the roads under study than in plants 200 m from the roads. References

4.7 Industrial surveys

Several studies have been undertaken in relation to heavy metal burdens and zones of influence with respect to industrial point sources. Trees and forests, particularly in association with forest decline, appear to be examined more frequently than other higher plants. Sampling stations are generally situated along transects at increasing distances from the point source in known wind directions.

In a study of heavy metal burden in air, soil and plants around a zinc smelter in India, Agrawal et al. (1988) did not observe statistically significant correlations between heavy metal content in the air, soil and plants. Local topography and microclimate of the study area also played a role in the dispersion of heavy metals. However, in general the zone of influence of the smelter extended to seven km. The leaves of the four plant species analysed (Mangifera spp., Acacia spp., Triticum spp. and Brassica spp.) showed great variation in heavy metal concentration depending on species, metal type and sampling site.

A variety of bioindicators were used to determine heavy metal fallout from a steel works in northern Finland along two sampling transects (Mukherjee and Nuorteva, 1994). Although the authors concluded that Hypogymnia physodes and Pleurozium schreberi were more appropriate bioindicators for most of the selected metals, the May-lily (Maianthemum bifolium) was a highly effective bioaccumulator of Cd from the iron and steel works. Other bioindicators under investigation included birch (Betula pubescens), Scots pine (Pinus sylvestris), Norway spruce (Picea abies) and Vaccinium sp. These showed generally higher Al, Fe and Zn concentrations in sampling areas within close vicinity to the steel works in comparison to sampling sites at a distance of > 6 km.

In another Finnish study, copper showed a pollution gradient along Scots pine (Pinus sylvestris) forest stands growing 0.5, 4 and 8 km from a copper-nickel smelter in Harjavalta (Helmisaari et al., 1995). Mean copper concentrations in one-year old pine needles in 1992 were 8.74, 20.04 and 210.53 mg/kg at distances of 8, 4 and 0.5 km from the smelter respectively. Analysis of Pinus sylvestris fine root samples implied that root uptake from the soil was the primary route of copper. Copper accumulation was significantly higher in the roots than in the leaves and copper concentrations in the leaves elevated significantly when the roots appeared to become saturated. This study demonstrated the significance of long-term heavy metal accumulation in the soil and its effects on soil processes and vegetation. The same smelter was investigated by Koricheva and Haukioja (1995) who discovered an exponential decrease in Cu, Ni, Fe and Zn concentrations in the leaves of Betula sp. with distance from the factory. References

In terms of point sources of air pollution and vegetation, less work has focused on incinerators than on other industrial emission sources. Elemental analysis of grasses in the vicinity of a municipal refuse incinerator in the United States found that concentrations of Cd, Fe, Hg, Mo, Pb and Zn were highest within 100 m of the incinerator (Bache et al., 1991). Of these metal species, all but Hg showed inverse relationships with logarithmic distance downwind. Hg showed a linear relationship with distance downwind. However, no soil or root analysis was undertaken during the survey, which makes it difficult to distinguish between the contribution of metals from root uptake and from direct aerial deposition. This study is of significance in that incinerators are often located in rural areas where contaminated vegetation may be used for pasture or edible crops.

The primary source of Hg in the aerial parts of a plant is, like Pb, generally thought to be via aerial deposition and not via translocation and root uptake (Zhang et al., 1995; Bache et al., 1991). Plant parts accumulate Hg from interception of wet and dry precipitation and absorption of gaseous Hg. References

4.8 Response methods

It has been observed that aerial heavy metal pollution has biochemical/ physiological effects on plants. Effects on growth have also been detected in response to air pollution. Many parameters can be affected by heavy metals. Toxic heavy metals may effect germination, young or old trees, stem growth, leaf formation, root growth, flowering/fruiting, plant growth rate and biomass, photosynthesis, transpiration, mineral nutrition and secondary metabolism etc. (Breckle and Kahle, 1992; Csintalan and Tuba, 1992). Some of these responses may be useful in the bioindication of aerial heavy metal contamination where elemental analysis proves too expensive or time consuming.

Most response techniques used in bioindication are in relation to gaseous pollutants but some emphasis has been placed on physiological responses with regard to metal pollutants. Most studies are in relation to industrial sites where a combination of air pollutants may be emitted. It is often difficult to determine if single aerial pollutants such as sulphur dioxide (SO₂) or a combination of pollutants including metals are more important in producing observed deleterious effects on plants. Effects may be additive, synergistic or antagonistic.

Examples of the limited number of studies which include plant responses to aerial metal depositions in the field are discussed briefly below. Most of these are controlled field study or in vitro experiments whereby seeds are sown in known contaminated areas or artificially enhanced contaminated locations. It is assumed that high levels of heavy metals in the soil medium will produce similar effects as atmospheric heavy metal pollution. Frequently such culture experiments are primarily aimed at determining threshold toxicity of pollutants to plants. References

In the industrial area of Shoubra Elkheima near Cairo, heavy metal contamination of soil and vegetation has occurred. In a study of the area, seeds of clover (Trifolium pratense) and Egyptian mallow (Malva parviflora) were sown in pots which were placed in nine fields at each of three sampling locations at increasing distances from the industrial pollution sources (Ali, 1993). A selection of injurious responses were observed in plants at the two locations within the vicinity of the industrial area in comparison to the control station. These include: a reduction in chlorophyll content; an increase in the number of plants with visible damage and in the area of injured leaves; a reduction in leaf number; a reduction in plant growth and weight. Much of this visible injury may be attributed to SO₂, NO_x and ozone pollution. However, Cd and Pb were present in the vegetative parts of the crops in elevated quantities and may be partly responsible for the observed effects. The lack of specificity of the aforementioned responses limits their utility as diagnostic tests of heavy metal contamination. According to Turcsanyi (1992), changes in chloroplast number and volume represent typical plant damage due to salts of heavy metals. The same author describes other cellular effects exerted by the salts of heavy metals.

In Slovakia, Kodrik (1994) measured root biomass and length in Norway spruce (Picea abies) at four sites of varying emission regimes. Although destructive, this method is appropriate as a measure of ecosystem health.

The combined effects of acidic and trace-elements on in vitro pollen germination and tube growth in a variety of higher plant species was investigated by Cox (1988). This acts as a potential bioindication method of the effect of air pollutants on reproduction processes.

Breckle and Kahle (1992) reported on a range of growth responses, mineral uptake and transpiration rates of beech seedlings to increasing exposure of Cd and Pb in isolation and in combination in the soil medium. Dose-response levels were comparable to metal concentration levels in German forests. Direct uptake of heavy metals through the leaf after deposition is an important route, especially for Pb, although this investigation was primarily concerned with effects of Pb and Cd on trees after root uptake. However, the measurable effects discussed in this paper, such as root elongation and architecture and leaf development, may still be appropriate in biomonitoring.

Toxic symptoms in response to Ni exposure to roots were displayed by cultured tomato plants under controlled experimental conditions in a study undertaken by Prokipcak and Ormrod (1986). The leaflets nearest the main stem appeared to be the most sensitive to Ni treatment. In a study of the response of a selection of crop plants transplanted to the vicinity of a copper smelter in Poland, older leaves were affected first (Fabiszewski et al., 1987). Typical Ni-injury in tomato plants was visible as interveinal chlorosis followed by necrosis distinguishable as 'small beige patches of dead tissue surrounded by darker brown or purple pigmented tissue'. It is noteworthy that chlorosis is often not specific and can vary with the time of year (Saxe, 1996). Decreases in growth response parameters such as leaf, stem and root weight, leaf area and plant height were also observed with increased Ni exposure to the roots. References

4.9 Transplants

Transplantation exercises are not as common using higher plants as they are utilising lichens and mosses. However, a limited demonstration of their use as transplants is provided below.

Pilegaard and Johnsen (1984) used transplantation to study the extent of heavy metal uptake by Achillea millefolium (milfoil) and Hordeum vulgare (barley) through their leaves and roots. Plants were grown in pots and exposed at areas of different aerial heavy metal deposition for 75 days prior to analysis.

Horse bean (Vicia faba minor), blue lupine (Lupinus angustifolius), oat (Avena sativa) and red fescue (Festuca rubra) were grown in a greenhouse prior to transplantation 800 m from a copper smelter for two weeks. Acute chlorosis associated with a reduction in chlorophyll-a and chlorophyll-b in leaves was significantly different from the control plants remaining in the greenhouse. Red fescue was the most sensitive species under investigation. References

II HEAVY METALS

4 Higher plants

4.1 Introduction