BIOLOGICAL MONITORS OF AIR POLLUTION

j . KUCERA
Czech Ecological Institute, NAA Laboratory,
CS-250 68 Rez near Prague, Czech Republic

Abstract

Direct biological monitoring of air pollution was introduced about 30 years ago. Although still
u.iduf development, the zppUcsticn of biologic;;! monitors, o r indicators, may provide important information
on the levels, availability, and pathways of a variety of pollutants including heavy metals and other toxic
trace elements in the air. A survey is given of the most frequently used biomonitors, such as herbaceous
plants, tree leaves or needles, bryophytes, and lichens, with their possible advantages and/or limitations.
In addition to using naturally-occurring biomonitors, a possibility of employing "transplanted" species in the
study areas, for instance grasses grown in special containers in standard soils or lichens transplanted with
their natural substrate to an exposition site, is also mentioned. Several sampling and washing procedures
are reported. The importance of employing nuclear analytical methods, especially instrumental neutron
activation analysis, for multielemental analysis of biomonitors as a pre-requisite for unlocking the
information contained in chemical composition of monitor's tissues, such as apportionment of emission
sources using multivariate statistical procedures, is also outlined.

1. THE BASIC CONCEPTS OF BIOLOGICAL MONITORING

Biomonitoring, i.e. the use of biological indicators to detect changes in the physical
and chemical properties of the abiotic environment, represents an interesting alternative
to direct measurements of the physical and chemical properties of the environment.
Biological indicators are those organisms (or populations) of which their occurrence, vitality
and responses change under the impact of environmental conditions [1]. Biomonitoring
takes one of t w o approaches:

i) direct monitoring which is based on measuring the quantity of pollutants in

suitable organisms rather than in samples from the environment;

ii) indirect monitoring which is based on interpreting of biological signals due

to changes of the environment such as the study of morphological,
physiological and cytological responses of organisms, changes of
abundances of certain species, etc.

Only the former approach will be treated in this report.

Although data provided by physical and chemical monitoring are indispensable for
evaluating the changes of the environment, the application of an (ideal) biomonitor can
show several advantages compared to the use of direct monitoring techniques [1-5].

the concentration of pollutants in the monitor organism are often higher than
in the system to be monitored. This may facilitate accurate sampling and

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 analysis, which are very difficult at the low levels occurring in many
compartments of the environment;

sampling of the organism used as a biological monitor is in general easier

than most direct sampling procedures and no long-term use of expensive
sampling equipment is required;

the intricate equilibria existing in many parts of the environment can easily
be distorted by sampi:ng itself, which may iead to erroneous results. When
using biological monitors, this distortion is minimized;

most organism reflect external conditions averaged over a certain time,

depending on e.g. the biological half life of a specific substance in that
organism. This is important when monitoring levels may change rapidly in
time;

concentrations of pollutants in organisms may give insight into the

bioavailability of that pollutant. This information may be as relevant as the
absolute concentration in a certain part of the environment;

biological monitors are already present in the environment and monitoring

continuously.

2. REQUIREMENTS ON BIOLOGICAL INDICATORS

In direct biomonitoring of atmospheric element pollution, the relevant information

is deduced from concentrations of elements in the monitor tissues. They have to meet
specific requirements which are as follows (6,71:

abundant occurrence in the area of interest, independent of local conditions;

available for sampling in all seasons;

tolerant to pollutants at relevant levels;

response to the quantity to be monitored known and understood;

element uptake independent of local conditions other than the levels of

elements to be monitored;

element uptake not influenced by regulating biological mechanisms or

synergistic effects;

averaging over suitable time period;

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 absence of appreciable element uptake from sources other than atmosphere;

low background concentrations;

easy sampling and sample preparation;

element accumulation to concentration levels accessible by routine analytical

techniques.

Many animal and plant species can be used for air pollution monitoring, because
they can meet most of the above requirements. Animal species and/or tissues usually
reflect complex changes of the environment, i.e. air, water, soil pollution, and element
intake from their diet, so that their use for studying only air pollution may be difficult to
interpret. Nevertheless, some of them were very popular in certain periods such as
analysis of human and/or animal hair. Certain plant species appear to be especially suitable
to indicate elemental air pollution and therefore advantages and pitfalls of their possible
use as biomonitors for air pollution studies will be of the main concern of this report.

3. HERBACEOUS PLANTS

Several naturally occurring plant species seem to be suitable accumulation

indicators of heavy metals. Most ruderal plants can be used for comparative evaluation
of air pollution owing to their high resistance to pollutants and common occurrence.
Different plant organs contain different amounts of elements. The highest quantities of
trace elements can usually be detected in roots and leaves, while stems and fruits (seeds)
contain lower amounts. Composition of leaves indicates both soil pollution (by subsequent
uptake of bioavailable pollutants by roots) and load from the air. The washing of plant
samples is a frequently debated issue. Obviously, in studies aiming at determining
endogenous element contents, the plant samples should be washed to eliminate the
external contamination. For samples to be employed for studying the air pollution load and
in so-called food chain studies, washing should be omitted. In the former case, a
standardized washing procedure (concerning a washing time and a washing agent) should
be employed.

The leaves of Lolium perenne and L. multiflorum have been reported to be suitable
biomonitors for many elements [1] and a positive correlation between the chemical
composition of the leaves of Taraxanum officiate and the local impact for the elements As,
Br, Cd, Co, Cr, Cu, Hg, Mn, Pb, Sb, Se, Zn has also been found [8,9]. Other plants have
been recognized especially suitable to indicate loading with selected elements, for instance
members of the genus Thlaspi for Ni and Zn [1], Solidago canadensis for F and Pb [10],
for vanadium Achilea milleforum, Amaranthus rotroflexus, Artemisia vulgaris,
Ca/amagrostis epigeios, Conysa canadensis, Echinochloa crus-galli, Plantago lanceolata
[11], while Solidago graminifolia, Trifolium pratense and Zea mayas are accumulation
indicators of organic pollutants, for example PCBs [12].

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 If a suitable plant does not occur in an area to be monitored, standardized cultivated
plant species grown in special pots (exposure containers) and standard soils can be
exposed at the site of an experiment for a definite period of time. Lolium multiflorum is
one of the indicators used most frequently for this purpose [1].

4. TREES

4.1. Broad-lea^eH trees and .shrubs

Tree leaves absorb elemental pollutants either from the air or from the soil by root
absorption. Thus, the same applies for their washing as for the leaves of herbaceous
plants. The chemical composition of leaves varies with their age. The element content,
especially of heavy metals, is generally higher at the end of summer; therefore this seems
to be the appropriate period for sampling. Many trees of general use for biomonitoring and
trees and shrubs specialized for indicating selected element at trace- and ultratrace levels
have been reported [1]. Two of the recommended trees seem to have found the widest
application as indicators of air pollution, mostly because of their resistance to pollutants
and wide distribution in Europe and almost throughout the world: Populus nigra ssp. italica
(Italian poplar) and Robinia pseudoacacia (the black locust tree) [1,10,13-16].

Sampling is recommended to be carried out in late August and early September

according to the following procedure: eight twigs are cut at a height of 5-8 m and of 2.5-3
m for the former and latter tree, respectively, three one-year-old shoots are cut from each
twig and leaves (preferably sun-leaves) from the middle of each shoot are collected [1].

Various pollutants of organic origin (e.g. PCBs) can also be indicated by the leaves
of Populus tremuloides and Rhus typhina [12].

4.2. Coniferous trees

Coniferous trees are more sensitive indicators of air pollution than the deciduous
trees, because they are exposed to air pollution over a longer period of time than leaves,
due to a longer life span of needles (3-4 years), except for Larix decidua which sheds all
its needles each year. Thus, most of the coniferous trees can respond to low pollutant
concentrations [ 1 ]. Taxusbaccata, Picea abies, PinussilvestrisandPseudotsugamenziesii
are the most frequently used indicators of elemental air pollution [1]. PCBs can also be
indicated by chemical analysis of Pinus strobus needles [12].

Similarly as with leaves, the chemical composition of needles of coniferous trees

is influenced by two factors:

i) by their nutritional status, i.e. by uptake of nutrients and/or pollutants by

roots from soil;

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 ii) by retention of air pollutants on the needle surface.

In the case of spruce needles, aerosol retention is facilitated by the very rugged
structure of epicuticular wax. The loading of spruce needles with solid, filterable aerosol
is about 1-2 mg/g dry needle weight. It can be removed completely from the needle
surface by washing either with CHCI3 or with a mixture of tetrahydrofuran/toluene (1:1,
vol./vol.) without influencing the inherent element concentrations in the needles [17].

Tree bark is also exposed to long-term air pollution and it accumulates certain elements
and sulphur dioxide H I .

5. BRYOPHYTES

Bryophytes are especially suitable for biological monitoring of air pollution due to
several specific features. They are evergreen and (with a few exceptions) perennial plants,
so that they can be utilized throughout the year. Many species have a wide geographical
distribution and grow in a wide range of habitats, which is beneficial for comparative
studies. Most bryophytes species do not posses a cuticle and, therefore, can take up
water over the entire plant surface. As a consequence, they obtain their nutrients directly
from atmospheric deposition, i.e. dustfall and precipitation. By comparing fresh specimen
with herbarium specimen we can also perform retrospective studies on elemental air
pollution. Retrospective investigations can also be performed by element determination
in peat profiles [18].

Ombrotropic peat bogs proved to be very useful indicators of long range

atmospheric transport of pollutants from Europe to Norway [19]. Their obvious
disadvantage is not very common occurrence.

Especially useful are some moss species that have a layered habit and produce
distinct annual segments. The most frequently used moss species are Hylocomium
splendens and Pleurozium schreberi which have the ability to accumulate many metals in
extremely high concentrations. For instance, in a highly polluted region in the vicinity of
a copper mine, a lead concentration of 17,320 //g/g was found in the moss H.splendens,
while the corresponding values in the Picea and Clinton/a species were 349.5 and 548.5
/yg/g, respectively [20].

Biomonitoring involving annual analyses of Hylocomium splendens and Pleurozium

schreberi has been included into the programme on Long-Range Transboundary Air
Pollution and the following sampling procedure has been recommended: Green (or
brownish green) shoots from the three most recent years are collected, excluding the
half-developed segment from the latest growing period. All dead material and attached
litter should be carefully removed and the samples are then dried at 40 °C to a constant
weight [21].

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6. LICHENS

Lichens are specialized organisms in which a fungus and an alga form a nutritional
and physiological unit. The autotrophic alga supplies nutrients for both itself and the
heterotrophic fungus. Lichens react to the pollutant emissions. Their high sensitivity to
various air pollutants - in comparison with higher plants - can be ascribed to both
morphological and physiological differences which namely include:

in the absence of a cuticle, pollutants find an easier way into the thallus;

corticolous lichens absorb both water and nutrients directly from the air;

lichens accumulate various materials without selection;

the material once absorbed will accumulate since there is no excretion.

About seventy lichen species have been reported as suitable indicators of elemental
pollution [1 ]. Of these, Hypogymnia physodes (L.) Nyl. and Parmelia sulcata are the most
frequently used species for biomonitoring in Europe. The former species is widespread in
Northern Europe and in mountain regions. It has many advantages which are important
for application as a biomonitoring organism, compared to other lichen species [22]:

it is one of the most tolerant lichen species, especially to S0 2 ;

it can withstand long periods with fairly low levels of humidity in the
atmosphere, hence its metabolic activities, including an active mechanism
for metal uptake, would be less affected by periods of low humidity than
would be the case with moisture-sensitive lichens;

this lichen has no rhizinae and this perhaps accounts for the insignificant
amounts of metals it absorbs from the substrate;

it has a large surface to weight ratio.

The enrichment factors (highest measured element concentration in the lichen from
polluted areas divided by corresponding values from control areas) for individual heavy
metals as measured in the environs of steel and iron works in Denmark showed the
following values [23]: Cd 74, Pb 62, Cu 35, Cr 34, V 3 1 , Fe 28, Mn 28, Zn 26, Ni 9. On
the other hand, Hypogymnia physodes grows on acidic bark substrates, which are less
suitable for monitoring purposes due to possible leaching of metals from the lichen and it
is less frequent in sub-Mediterranean areas. In these areas, as well as the European plains,
Parmelia species are more frequent and investigated [24]. Lichens may also show
interfering element uptake from the substrate, but are more widespread than mosses,
especially in densely populated or industrialized areas. Furthermore, substrate/soil
interferences can be recognized by the application of factor analysis techniques [24-26].

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Removing adhering sand and/or soil from the lichens is also a debated issue. Washing with
distilled water followed by oven drying at 30 °C was used at the beginning of a national
Dutch study [26], but this practice has stopped in a later stage [27].

Another problem of utilizing lichens for monitoring air pollution may be due to stem
flow which may bring to lichens additional nutrients and/or pollutants not necessarily
associated with air pollution as depicted in Fig.1 [21]. This problem can be avoided by
employing lichens in the form of "transplantedncolonies (growing on their natural substrate
pieced or: en exposure plate - Cf. Fig.2) according to the internationally accepted, standard
method [28]. In this way, biomonitoring may be used even in areas where lichens do not
occur. Parmelia (hypogymnia) physodes is most frequently used for this purpose, but the
following species are also suitable for transplantation: Parmelia sulcata, P.caperata,
P.cortea, P.furfuracea, Xanthoria parietina, Evernia prunastri. The transplanted lichens
indicate the level of pollution within a short time (4-15 weeks).

Figure 1. Schematic illustration of fluxes in an ecosystem [21]

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 Lichen colony
on a disk of
natural
substrate

Figure 2. Exposition table for lichen species [1]

7. CONCLUSIONS

The use of biological indicators for air pollution monitoring was introduced about
30 years ago. Since then, a variety of organisms have been proposed for biomonitoring
purposes. Most of them have been enumerated in a recent book [1], however, without
their critical evaluation. The importance of particular biomonitors can be inferred from the
frequency of their use in various national and international programmes. For instance, the
following biomonitors of air pollution are being collected for the German Environmental
Specimen Bank: grass, poplar and beech leaves, spruce and pine shoots [29]. Grasses and
other herbaceous plants may be useful indicators, especially if they are grown on standard
soil in exposure containers (pots) [30]. Mosses have been very popular in both local and
long-term, large-scale studies in Nordic countries since seventies [19,31] and using these
bioindicators have also been included in the LRTAP programme [21]. In the Netherlands,
a national trace-element air pollution monitoring survey using epiphytic lichens has been
started in 1982 [24-26] and continues on international scale until now [27]. In the Dutch
studies [24-26], lichen analysis followed by a specific multivariate statistical procedure
known as " Target Transformation Factor Analysis" has successfully been employed for
apportionment of emission sources inside and outside the country. To obtain the relevant
information of this kind, multielement analysis is required. For this purpose, nuclear
analytical methods, namely instrumental neutron activation analysis, proved to be very
effective tools. On the other hand, for all the biomonitors employed, the mechanisms of

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pollutant uptake and retention are still not sufficiently known due to intricate element and
water fluxes in an ecosystem, as illustrated in Fig.1. Consequently, the quantitative
relationships between the pollutant's concentration in the monitor's tissue and its
concentration in a relevant compartment of the atmosphere are not predictable. Therefore,
without extensive calibration under all relevant conditions, the use of even of one of the
most suitable biomonitor will yield only qualitative information on atmospheric levels as a
function of time or place [ 3 2 ] .

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