BIO 408

PLANTS AND ENVIRONMENTAL POLLUTION MONITORING (3 UNITS)

O. OYETUNJI (PhD)
Course contents
Introduction to environmental pollution
Causes of environmental pollution
The use of algae, lichen, bryophytes and higher plants in monitoring environmental pollution.
The use of algae as indicators of aquatic pollution.
The merits and demerits of using various taxonomic groups as indicators.

Introduction to environmental pollution

Environmental pollution is defined as "the contamination of the physical and biological
components of the earth/atmosphere system to such an extent that normal environmental processes
are adversely affected.

Air is an important and vital component of this planet and any slight change in its composition
may have negative effects on the growth, development and survival of different organisms on this
planet. Air pollution refers to the number of harmful substances in the atmosphere of concentration
and residence time more than the allowed range, which is beyond the capability of diffusion and
dilution resulting in air quality deterioration, brought bad influence directly or indirectly to human
health and ecological environment. It is categorized into gaseous (mainly SO2, NOx and O3) and
dust particles which include some heavy metals. It is one of the severe problems facing the world
today due to the continual change in concentration levels of some gaseous and trace metals in the
environment resulting from man’s activities such as road transportation, vehicular traffic and
industries. Air pollution has become a major environmental risk as far as public health is
concerned. Reducing levels of air pollution may also reduce the global burden of disease. W.H.O
has estimated that approximately 2 million and 1.3 million deaths worldwide mostly in developing
countries have occurred due to indoor and outdoor air pollution. Several plants are known to be
susceptible to very low concentrations of air pollution and exhibit a characteristic foliar injury
following exposure to a specific air pollutant. They can therefore be used to determine the presence
of air pollution in a given area.
Monitoring for injurious levels of air pollutants by plants is a standard technique used in the
diagnosis of air pollution injury on plants. In recent years, increasing efforts are being made to use
plants for detection of air quality.
Biomonitoring is generally defined as the systematic use of living organisms or their responses
to determine the condition or changes in the environment. Biological monitoring of air pollutants
can be passive or active. Passive methods observe plants growing naturally within the area of
interest. Active methods detect the presence of air pollutants by placing test plants of known
response and genotype into the study area.

Biomonitoring includes four concepts; the use of biomarkers, bioindication, biointegration and
bioaccumulation. Monitoring of air pollution using plants is cost effective and environmentally
friendly technique that substitute physical and chemical analytical methods of air pollution
monitoring systems which is costly and unfriendly to the environment.

Categories of contaminants
The compounds released into the environment as a consequence of human activities can be
classified in two main categories:
1) Biodegradable substances and
2) Conservative substances.

Notable Pollutants Notable Pollutants include the following groups:

1. Heavy metals
2. Persistent organic pollutants (POP)
3. Environmental persistent pharmaceutical pollutants (EPPP)
4. Polycyclic aromatic hydrocarbons
5. Volatile organic compounds
6. Environmental xenobiotics

Different Types of Pollutants by Absorptive Capacity

Stock pollutants – Pollutants that the environment has little or no absorptive capacity is called
stock pollutants e.g. persistent synthetic chemicals, non-biodegradable plastics, and heavy metals.
Stock pollutants accumulate in the environment over time. The damage they cause increases as
more pollutant is emitted, and persists as the pollutant accumulates. Stock pollutant can create a
burden for future generations by passing on damage that persists well after the benefits received
from incurring that damage have been forgotten.

Fund pollutants – are those for which the environments have some absorptive capacity. Fund
pollutants do not cause damage to the environment unless the emission rate exceeds the receiving
environment‘s capacity (e.g. carbon dioxide, which is absorbed by plants and oceans). Fund
pollutants are not destroyed, but rather converted into less harmful substances, or diluted/dispersed
to nonharmful concentrations.

Primary Pollutants
Primary pollutants are pollutants that are directly emitted from a process, such as ash from a
volcanic eruption, the carbon monoxide gas from a motor vehicle exhaust or sulphur dioxide
released from factories. Major primary pollutants include:
 Sulphur Oxides (SOx) – especially sulphur dioxide, a chemical compound with the fomula SO2
is produced by volcanoes and in various industrial processes. Since coal and petroleum often
contain sulphur compounds, their combustion generates sulphur dioxide. Further oxidation of SO2,
usually in the presence of a catalyst such as NO2, forms H2SO4,and thus acid rain. This is one of
the causes of concern over the environmental impact of the use of these fuels as power sources.
 Nitrogen Oxides (NOx) – especially nitrogen dioxide are emitted from high temperature
combustion, and are also produced naturally during thunderstorms by electrical discharge. Can be
seen as the brown haze dome above or plume downwind of cities. Nitrogen dioxide is the chemical
compound with the formula NO2. It is one of the several nitrogen oxides. This reddish- brown
toxic gas has a characteristic sharp, biting odour. NO2 is one of the most prominent air pollutants.
 Carbon monoxide (CO) – is colourless, odourless, non-irritating but very poisonous gas. It is a
product by incomplete combustion of fuel such as natural gas, coal or wood. Vehicular exhaust is
a major source of carbon monoxide.
 Carbon dioxide (CO2) – a colourless, odourless, non-toxic greenhouse gas also associated with
ocean acidification, emitted from sources such as combustion, cement production and respiration.
It is otherwise recycled in the atmosphere in the carbon cycle.
Secondary Pollutants
These are not emitted directly. Rather, they form in the environment when primary pollutants react
or interact. An important example of a secondary pollutant is ground level ozone – one of the many
secondary pollutants that make up photochemical smog. Some pollutants may be both primary and
secondary, that is, they are both emitted directly and formed from other pollutants.
 Secondary pollutants include particulate matter formed from gaseous primary pollutants and
compounds in photochemical smog. Smog is a kind of air pollution; the word ‗‘Smog‘‘ is a
portmanteau of smoke and fog. Classic smog results from large amounts of coal burning in an area
caused by a mixture of smoke and sulphur dioxide. Modern smog does not usually come from coal
but from vehicular and industrial emissions that are acted on in the atmosphere by ultraviolet light
from the sun to form secondary pollutants that also combine with the primary emissions to form
photochemical smog.
 Ground level ozone (O3) formed from NOx and VOCs. Ozone (O3) is a key constituent of the
troposphere. It is also an important constituent of certain regions of the stratosphere commonly
known as the ozone layer. Photochemical and chemical reactions involving ozone drive many of
the chemical processes that occur in the atmosphere by day and by night. At abnormally high
concentrations brought about by human activities (largely the combustion of fossil fuel), it is a
pollutant, and a constituent of smog.
 Peroxyacetyl nitrate (PAN) – similarly formed from NOx and VOCs.

Causes of pollution
Biodegradable substances represent the larger volume of human-generated wastes (domestic
wastes, industrial wastes, etc.) and, depending on their origin, they are disposed of in the air, the
soil, or water. They consist of organic material (rich in C, N, and P) liable to bacterial degradation
through oxidative processes that reduce these organic compounds to soluble inorganic compounds
(CO2, H2O, and H3N). If the release of these compounds is very high, anaerobic activity occurs,
with ensuing origination of degradation products (H2S, HN, H4C) that not only give off unpleasant
smells, but are also toxic for many organisms. Generally, uncontrolled release of biodegradable
waste products, especially into water bodies, causes eutrophication phenomena and subsequently
a decrease in the quantity of oxygen present in the medium in question; it can also bring about the
production of toxic degradation compounds. Conservative substances are not decomposed by
bacteria or other short-term processes. These substances are typically very reactive toward plants
and animals, sometimes causing considerable damages.
There are three main categories:
1. Heavy metals (Pb, Cd, Hg, Cr, Cu, Zn …);
2. Halogenated hydrocarbons (HHC), dichlorodiphenyltrichloroethane (DDT), and
polychlorinated biphenyls (PCBs);
3. Radioactive compounds –electromagnective waves etc

Goals of Biological Monitoring

Biomonitoring is the measurement of the body burden of toxic chemical compounds, elements, or
their metabolites, in biological substances. Biological monitoring can be divided into:
(a) Monitoring of exposure, and
(b) Monitoring of effect, for which indicators of internal dose and of effect are used respectively

The purpose of biological monitoring of exposure is to assess health risk through the evaluation of
internal dose, achieving an estimate of the biologically active body burden of the chemical in
question. Its rationale is to ensure that worker exposure does not reach levels capable of eliciting
adverse effects. An effect is termed “adverse” if there is an impairment of functional capacity, a
decreased ability to compensate for additional stress, a decreased ability to maintain homeostasis
(a stable state of equilibrium), or an enhanced susceptibility to other environmental influences.

Depending on the chemical and the analysed biological parameter, the term internal dose may have
different meanings (Bernard and Lauwerys 1987). First, it may mean the amount of a chemical
recently absorbed, for example, during a single workshift. A determination of the pollutant’s
concentration in alveolar air or in the blood may be made during the workshift itself, or as late as
the next day (samples of blood or alveolar air may be taken up to 16 hours after the end of the
exposure period). Second, in the case that the chemical has a long biological half-life—for
example, metals in the bloodstream—the internal dose could reflect the amount absorbed over a
period of a few months.
Third, the term may also mean the amount of chemical stored. In this case it represents an indicator
of accumulation which can provide an estimate of the concentration of the chemical in organs
and/or tissues from which, once deposited, it is only slowly released. For example, measurements
of DDT or PCB in blood could provide such an estimate.

Finally, an internal dose value may indicate the quantity of the chemical at the site where it exerts
its effects, thus providing information about the biologically effective dose. One of the most
promising and important uses of this capability, for example, is the determination of adducts
formed by toxic chemicals with protein in haemoglobin or with DNA.
Biological monitoring of effects is aimed at identifying early and reversible alterations which
develop in the critical organ, and which, at the same time, can identify individuals with signs of
adverse health effects. In this sense, biological monitoring of effects represents the principal tool
for the health surveillance of workers.

Biological monitoring of effects is aimed at identifying early and reversible alterations which
develop in the critical organ, and which, at the same time, can identify individuals with signs of
adverse health effects. In this sense, biological monitoring of effects represents the principal tool
for the health surveillance of workers.

Monitoring Methods

Biological monitoring of exposure is based on the determination of indicators of internal dose by

measuring:

 The amount of the chemical, to which the biological agents (human/ nonhuman) is
exposed, in blood or urine (rarely in milk, saliva, or fat, sap, juice)
 The amount of one or more metabolites of the chemical involved in the same body
fluids
 The concentration of volatile organic compounds (solvents) in alveolar air/ or stomata
 The biologically effective dose of compounds which have formed adducts to DNA or
other large molecules and which thus have a potential genotoxic effect
Unfortunately, there are only list of a few examples of the application of this approach, namely

(1) The inhibition of pseudocholinesterase by organophosphate insecticides,

(2) The inhibition of δ-aminolaevulinic acid dehydratase (ALA-D) by inorganic lead, and

(3) The increased urinary excretion of d-glucaric acid and porphyrins in subjects exposed to
chemicals inducing microsomal enzymes and/or to porphyrogenic agents (e.g., chlorinated
hydrocarbons)
Advantages and Limitations of Biological Monitoring
For substances that exert their toxicity after entering the human organism, biological monitoring
provides a more focused and targeted assessment of health risk than does environmental
monitoring. A biological parameter reflecting the internal dose brings us one step closer to
understanding systemic adverse effects than does any environmental measurement.

Biological monitoring offers numerous advantages over environmental monitoring and in

particular permits assessment of:

· Exposure over an extended time period

· Exposure as a result of worker mobility in the working environment

· Absorption of a substance via various routes, including the skin

· Overall exposure as a result of different sources of pollution, both occupational
and non-occupational

· The quantity of a substance absorbed by the subject depending on factors other than the
degree of exposure, such as the physical effort required by the job, ventilation, or climate

· The quantity of a substance absorbed by a subject depending on individual factors that can
influence the toxicokinetics of the toxic agent in the organism; for example, age, sex, genetic
features, or functional state of the organs where the toxic substance undergoes biotransformation
and elimination.

In spite of these advantages, biological monitoring still suffers today from considerable limitations,
the most significant of which are the following:

· The number of possible substances which can be monitored biologically is at present still rather
small.

· In the case of acute exposure, biological monitoring supplies useful information only for
exposure to substances that are rapidly metabolized, for example, aromatic solvents.
· The significance of biological indicators has not been clearly defined; for example, it is not
always known whether the levels of a substance measured on biological material reflect current or
cumulative exposure (e.g., urinary cadmium and mercury).

Relationship between exposure, internal dose and effects

The study of the concentration of a substance in the working environment and the simultaneous
determination of the indicators of dose and effect in exposed subjects allows information to be
obtained on the relationship between occupational exposure and the concentration of the substance
in biological samples, and between the latter and the early effects of exposure.

Knowledge of the relationships between the dose of a substance and the effect it produces is an
essential requirement if a programme of biological monitoring is to be put into effect. The
evaluation of this dose-effect relationship is based on the analysis of the degree of association
existing between the indicator of dose and the indicator of effect and on the study of the
quantitative variations of the indicator of effect with every variation of indicator of dose. (See also
the chapter Toxicology, for further discussion of dose-related relationships).

With the study of the dose-effect relationship it is possible to identify the concentration of the toxic
substance at which the indicator of effect exceeds the values currently considered not harmful.
Furthermore, in this way it may also be possible to examine what the no-effect level might be.

Since not all the individuals of a group react in the same manner, it is necessary to examine the
dose-response relationship, in other words, to study how the group responds to exposure by
evaluating the appearance of the effect compared to the internal dose. The term response denotes
the percentage of subjects in the group who show a specific quantitative variation of an effect
indicator at each dose level.

Applications of Biological Monitoring

The practical application of a biological monitoring programme requires information on (1) the
behaviour of the indicators used in relation to exposure, especially those relating to degree,
continuity and duration of exposure, (2) the time interval between end of exposure and
measurement of the indicators, and (3) all physiological and pathological factors other than
exposure that can alter the indicator levels.

In the following articles the behaviour of a number of biological indicators of dose and effect that
are used for monitoring occupational exposure to substances widely used in industry will be
presented. The practical usefulness and limits will be assessed for each substance, with particular
emphasis on time of sampling and interfering factors. Such considerations will be helpful in
establishing criteria for selecting a biological test.

Time of sampling
In selecting the time of sampling, the different kinetic aspects of the chemical must be kept in
mind; in particular it is essential to know how the substance is absorbed via the lung, the
gastrointestinal tract and the skin, subsequently distributed to the different compartments of the
body, biotransformed, and finally eliminated. It is also important to know whether the chemical
may accumulate in the body.
With respect to exposure to organic substances, the collection time of biological samples becomes
all the more important in view of the different velocity of the metabolic processes involved and
consequently the more or less rapid excretion of the absorbed dose.

Interfering Factors
Correct use of biological indicators requires a thorough knowledge of those factors which,
although independent of exposure, may nevertheless affect the biological indicator levels. The
following are the most important types of interfering factors.

Physiological factors including diet, sex and age, for example, can affect results. Consumption of
fish and crustaceans may increase the levels of urinary arsenic and blood mercury. In female
subjects with the same lead blood levels as males, the erythrocyte protoporphyrin values are
significantly higher compared to those of male subjects. The levels of urinary cadmium increase
with age.
Among the personal habits that can distort indicator levels, smoking and alcohol consumption are
particularly important. Smoking may cause direct absorption of substances naturally present in
tobacco leaves (e.g., cadmium), or of pollutants present in the working environment that have been
deposited on the cigarettes (e.g., lead), or of combustion products (e.g., carbon monoxide).
Metals are considered as one of the most hazardous pollutants in the environment due to their
persistence in the environment, bioaccumulation, and high toxicity. The source of metals in the
coastal area of the Black Sea is from anthropogenic activity, such as wastewater outflows,
industrial sludge, river discharges, airborne inputs, rainfall, and dust precipitation. The
concentration of metals in water is low and varies depending on environmental factors. Metal
concentrations in the sediment depend on the oxidation-reduction potential, organic content, pH,
and the grain size composition. The pollution levels of the aquatic environment by metals can be
estimated by analyzing water, sediments, and marine organisms. Marine organisms are one of the
most reliable indicators for identifying sources of biologically available metal contaminations. The
biomonitoring process has been widely used to establish the contamination with metals in the last
two decades. Different types of organisms may be used for biomonitoring, for example, marine
algae and filter-feeding molluscs. Many studies showed that biovalves did not regulate the level
of some metals within their body and the level of accumulated metals depends on the
contamination of surrounding areas. Seagrass and algae can also be used as bioindicators and give
information on concentrations of metals or bioavailability of metals in the surrounding
environment

Principles for bioindication/biomonitoring of the environment

Organisms, populations, biocoenoses and ultimately whole ecosystems are naturally influenced by
numerous biotic and abiotic stress factors such as fluctuations in climate, varying radiation and
food supply, predator-prey relationships, parasites, diseases, and competition within and between
species. This stress situation is vital at every level of biological organisation. Consequently, the
ability to react to stressors is an important characteristic of all living systems, and conversely no
development of the species and the ecosystem as a whole is possible without such natural stressors.
Through human activity the environment has been confronted with totally new substances that did
not previously exist (xenobiotics, many radionuclides) and potentially harmful substances released
in quantities unthinkable in the past (heavy metals, natural radionuclides).
In the last 200 years the increase in the world's population and the resulting global rise in energy
consumption have led to a dramatic change in the natural basis of our lives. Over 70 square miles
of arable land are being irreversibly transformed into desert through mismanagement, overgrazing
and over-population. Ten to 100 species of plants and animals may disappear from the planet each
day, and an even bigger problem is that we do not know whether the number is 10 or 100. N or we
do know whether the total number of species on earth is one million or 10 million (Schneider,
1992). Each day we will add 2700 more tons of chloro- fluorocarbons and 51 million tons of carbon
dioxide to our atmosphere and the earth will be a little warmer and a little more acidic, and more
ultraviolet radiation will pass through our atmosphere to the surface of the earth (Schneider, 1992).
A large amount of (chlorinated) pesticides will be used in the developed countries, and an even
greater but unknown amount of such compounds will be applied in the less developed countries of
the world.
Chemical substances constitute the greatest pollutant burden on natural ecosystems. The number
of species world-wide is thought to be 13 million, although only about 1.6 million species have
been identified. Pollution, habitat fragmentation and loss, intensification of agriculture and
population pressure are leading to dramatic changes in biodiversity.
Sustainability describes the worldwide goal of all future efforts towards development. It chiefly
means lastingly preventing the over-exploitation of:
 natural resources, especially soil and water;
 genetic diversity and the functional stability of the climate;
 the social basis for a livelihood and chances of development, especially in the poor sections
of the population,
USE OF ALGAE IN AQUATIC POLLUTION
Algae are ideally suited for water quality assessment because they have rapid reproduction rates
and very short life cycles, making them valuable indicators of short-term impacts. Algal
assemblages are typically species rich, and algal species exhibit wider distributions among
ecosystems and geographical regions. As primary producers, algae are most directly affected by
physical and chemical factors. Algal assemblages are sensitive to some pollutants and they readily
accumulate pollutants, and algal metabolism is also sensitive to the variation of environmental and
natural disturbances. Algae are easily cultured in the laboratory and sampling is easy, inexpensive
and creates minimal impact on resident biota; relatively standard methods exist for the evaluation
of functional and non-taxonomic structural characteristics of algal communities.
Periphyton are one of the most important algae associated with substrates in aquatic habitats.
Periphyton have been widely used as a tool for biologically monitoring water quality. These
organisms exhibit high diversity and are a major component in energy flow and nutrient cycling
in aquatic ecosystems. Many characteristics of periphyton community structure and function can
be used to develop indicators of ecological conditions in the aquatic ecosystem. Periphyton are
sensitive to many environmental conditions, which can be detected by changes in species
composition, cell density, ash free dry mass (AFDM), chlorophyll, and enzyme activity (e.g.,
alkaline and acid phosphatase). Each of these characteristics may be used, singly or in aggregation,
to assess conditions with respect to societal values, such as biological integrity and trophic
condition. The advantages that periphyton communities have over other organisms for monitoring
purposes include the following: fixed habitats, so they cannot avoid pollution; relatively quick
recolonisation after perturbations in water quality or flow, the ability to enable a rapid resumption
of monitoring; the ease of sample preparation for analysis; and widespread, common taxa, enabling
their pollution tolerances to become well known.
Diatoms have been used extensively in water quality monitoring. They exist in a wide range of
ecological conditions, colonising almost all suitable habitats; they can thus provide multiple
indicators of environmental change. Indices of water quality using diatoms gave the most precise
data compared to chemical and zoological assessment.

ALGAE AS BIOINDICATORS OF AQUATIC ECOSYSTEM

Aquatic populations are impacted by anthropogenic stress, resulting in a variety of alterations in
the biological integrity of aquatic systems. Algae can serve as an indicator of the degree of
deterioration of water quality, and many algal indicators have been used to assess environmental
status. Kolkwitz and Marsson in 1908 were the pioneers who classified algal species based on their
tolerance to various kinds of pollution. They stated that the presence of certain species of algae
could define various zones of degradation in a river. Diatom flora as a powerful indicator of
environmental change and its emergence as a preferred indicator in monitoring studies. Algae are
also used in laboratory bioassays to study water quality, using media for culturing indicator species
from the field or defined media to which varying degrees or concentrations of the pollutant are
added the algal attributes and indicators that could be used in biological monitoring programs. To
define the effects of various types of river degradation, it is important to use a variety of algal
parameters.

Approaches for Biomonitoring of Aquatic Ecosystems Based on Algae

The oldest approach for using algae to assess stream water quality is based on the indicator species
concept (Saprobien system). The Saprobien system is widely used in municipal and wastewater
monitoring and discriminates between polluted and clean streams. In monitoring study conducted
at a River Basin, the saprobic index, which was based on diatom species assemblages on glass
slides, successfully divided the sampling stations into four zones of saprobic contamination:
1. Polysaprobic Zone,
2. Alpha-mesosaprobic Zone,
3. Beta-mesosaprobic Zone and
4. Oligosaprobic Zone.

The zones of saprobic contamination were characterised by the occurrence of certain groups of
diatom species, namely
 Saprobiontic species,
 Saprophilic species,
 Saproxenous species and
 Saprophobous species.
The saprobic index was calculated based on periphytic algal species composition in the River
Basin, and revealed that water quality evaluation using diatom indices was consistent with the
physical and chemical determination
Algae sample will be collected manually in the tidal zone in the environment. There are different
types of algae eg
Cryptogamia class,
Cystoseira genus, and
Ulva algae
Different types of algae found in the area should be collected. The Samples collection should be
done between the hour of 10 am and 12 pm, which generally corresponded to rising daily
temperature and solar radiation conditions. The selected samples should be collected at a similar
stage of development. After washing (in the place of seawater), the samples will be rinsed in
deionized water in order to avoid losses of metal during treatment. The algae samples will then be
dried at 60 °C to constant mass and ground in an agate mortar. Samples of the top layer of sand
(0–20 cm layer) will be taken from several selected measuring positions located along the Black
Sea coast. Take water-dried sand and pass through a 1 mm sieve to remove stones and root
residues. All solutions should be prepared in high purity water obtained by reverse osmosis in a
Watek-Demiwa 5 Rosa system (Czech Republic), followed by a triple distillation from a quartz
apparatus. Only freshly distilled water should be used.

Instrumentation -A μAutolab electrochemical analyzer from EcoChemie (Utrecht, Netherlands)

or similar equipment should be used for electrochemical measurements. A standard three-electrode
configuration, consisting of a mercury film electrode based on glassy carbon (which can be used
as a working electrode), Ag/AgCl (3 M KCl) reference, and Pt wire counter electrodes, should be
incorporated into a glass cell (V = 20 cm3 ). The metal ions concentrations should be measured
using atomic absorption spectrometry (AAS) with a Z-8200 spectrometer equipped with premix
fishtail type burner – air/acetylene and NO2/acetylene, graphite furnace – flame, and furnace on
the same beam made by Hitachi, Japan.

Solutions and Preparations Standard solutions –Solution of Tl and other analyzed metals
should be prepared by dilution of a 1000-μg ml−1 stock standard solution obtained from Sigma
Aldrich. Ammonia solution (25%), nitric acid (65%), hydrofluoric acid (73%), hydrogen peroxide
(30%), EDTA, and ascorbic acid (supplied by Sigma Aldrich) should be used to conduct the
determination.
Determination of Thallium in the Algae Samples by the Voltammetric Method -
Determination of thallium in the algae samples should be conducted. Samples of the studied algae
(0.25 g) should be placed in a teflon beaker and digested by adding 65% nitric acid and 2.5 cm3
of 30% hydrogen peroxide. Upon evaporation of the solution, the residues should be mixed with
an additional dose of nitric acid (1 cm3), covered with glass, and heated for 3 h. After filtration,
the residues should be mixed with ascorbic acid (2.5 cm3 of 1 M solution) and EDTA (6.25 cm3
of 0.2 M solution). The pH of the solution could then be adjusted to a value of 4.5 (using an
ammonium solution), then it should be transferred to a flask (25 cm3) and supplemented with
water. The final solution should be used for the determination of thallium in the samples using
differential pulse anodic stripping voltammetry (DPASV). The pre-concentration of Tl should be
carried out at a potential of − 900 mV vs. SCE over 900–3600 s depending on the Tl concentration.
Voltammograms should be recorded after medium exchange for pure 0.05 M EDTA. The results
should be evaluated based on several additions of an internal standard (typically 3 additions). The
detection limit of the method (calculated on a 3SD basis) could be 50 pg L−1 (0.25 pM).
Determination of Metals in the Algae Samples by AAS -The algae samples (0.25 g) should be
digested in teflon vessels for 2 h with a mixture of 65% HNO3 and 73% HF acids, respectively,
according to the method described by Lukaszewski et al. (2012). The metals (Fe, Cd, Pb, Zn, and
Cu) should be analyzed using flame-atomic absorption spectrophotometer (AAS). Several
potential causes of the presence of trace elements in the algae and sand can be distinguished from
the analysis.

LICHEN AS BIOINDICATOR
Lichen can also be used to measure toxic elemental pollutants and radioactive metals because they
bind these substances in their fungal threads where they concentrate them over time.
Environmental scientists can then evaluate this accumulation to determine the history of the local
air.
A lichen is an associated organism: two very different beings, an alga and a fungus, live together
in a qualified symbiotic association, producing a new body, or lichen thallus. Much of the lichen
body is a tangle of fungal filaments called hyphae; these filaments clasp alga, sometimes in a mat,
or sometimes wrapped as single cells. The fungus collects water and provides structure and
protection for the alga, and may in certain species extract minerals for both organisms from the
substrate. The alga possesses chloroplasts and can photosynthesize, thus providing carbohydrates
for both itself and its fungal partner. When a fungus joins with an alga, it is said to be "lichenized."
Many scientists, consulting a family tree of DNA, now believe that different forms of lichens
evolved in independent strands of fungi development. They do not agree on the number of strands.
Lichens have special adaptations which permit them to withstand extremes of moisture and
temperature. When moisture is available, it is taken up by the fungus leading to a mechanical
change which allows more light to get through, triggering algal photosynthesis; new food and new
tissue are then made. When the atmosphere is dry, however, the lichen is dormant and does not
grow. Because lichens are hardy, love light, live a long time, and grow slowly, they can be
pioneers in many inhospitable environments such as tundra, exposed rock surfaces, asbestos,
mortar, tropical leaf surfaces, and even in water. When they decay, their nutrients nourish new
settlers such as moss.
Types of lichen
Crustose (crusty)
Crustose lichens form a “crust” onto their substrate of trees, rocks or soil. The crust is attached
so firmly that it cannot be removed without causing damage. They possess:
 edges flat, unlobed and closely attached to substrate
 hard to remove without damaging substrate or lichen
 algae usually dispersed
 edges unlobed (leprose and squamulose included here)

Foliose (leafy)
Foliose lichens are leafy (think: foliage) that attach loosely, and the lobes of the leaf are often
parallel to the surface of the substrate. They possess:
 A sandwich of fungal layer with algal mat in middle
 circular growth, lobes
 small rootlets called rhizines attach it to substrate
 top and bottom layers different

Foliose lichen

Fructicose (shrubby)
Fruticose lichens are the three dimensional, often growing perpendicular to their substrate. They
can look like little bushes growing off the side of a tree or rock. They possess:
 round branches with its fungal layer outside, its algal layer within
 no rhizines
 vertical growth pattern
 odd-shaped structures such as globets threads
Fruticose lichens are either shrub-like small mounds, growing up from the ground, or beard-like,
small tangles looking a bit like spanish moss when hanging down, attached to the substrate only
at their bases, and usually with a circular cross-section. In addition, Leprose lichens are powdery
masses with little or no organized structure; Squamulose are much the same as crustose, but have
raised edges, which can be folded and lobe-like. All but fruticose lichens grow slowly; their
growth, about .5 to 5 mm per year measured by the expansion of their circles. Fruticose lichens,
on the other hand grow vertically, and quickly, up to 2 cm per year. Left unchallenged, undisturbed
and with a suitably long-lived substrate, it is quite common for a lichen to have a lifespan of several
centuries; in fact a certain arctic specimen of a crustose lichen, Rhisocarpon geographicum, was
found to be about 9000 years old.

Lichens and Nitrogen

Lichens are sensitive to atmospheric pollution such as nitrogen (N) because they receive all their
nutrients and water from wet and dry atmospheric deposition (fall out). Nitrogen deposition can
increase the load of nutrients. Too much N can harm and kill the algae’s chlorophyll which is used
to produce sugars feeding it and the fungi.
Certain species of lichen are more tolerant of N than others. Scientists monitor lichen communities.
If an increase in N tolerant species in combination with a decrease in N sensitive species occurs
this may indicate an increase in atmospheric N deposition.
Lichens are the “canaries in the coal mine” of N deposition. A shift in their species composition
and/or their health exemplifies the potential beginning of ecosystem decline due to N deposition.

Effects of SO2 on Lichens

Lichens are injured by sulfur dioxide (SO2). Rose (1975) has calculated that more than one-third
of England and Wales has lost nearly all its epiphitic lichens, the most delicate shrubby lichens,
largely due to the sulphur-dioxide emissions of coal-burning power plants. In Northern Siberia, an
area of the Soviet Union which is very polluted, the number of lichen species has fallen from 50
to about 3, and the lichen production in general stands at about 1 or 2% or normal levels,
threatening the reindeer diet; in Alaska there are similar concerns about lichen reduction and the
caribou diet. (Tyson, 1990). Losses in other parts of the world reflect the increasingly poor quality
of the earth's air and the need for early warning bioindicators such as lichens. This pollutant has
natural sources, such as volcanic eruptions and sea spray. By far the largest source for it, however,
is the combustion of fossil fuels, automobile emissions, and some industrial processes. The
pollutant is carried in the atmosphere until rained out or deposited as dry particles or as gas. Sulfur
dioxide combines with moisture in the atmosphere to form sulfurous acid (H2SO3) or sulfuric acid
(H2SO4). When this happens with rainwater, the result is acid rain. All these forms of sulfur are
harmful to lichens and plants.
Lichens have also shown sensitivity to some other pollutants, such as heavy metals and ozone, but
for the most part lichen damage can be attributed to SO2. The effect of pollution upon lichen
depends on the pH of the substrate, the surface on which the lichen grows. In general, an alkaline
substrate such as basic bark or limestone counteracts the acidity of SO2 pollution. As acid rain falls
on a substrate, one kind of lichen growth form will often be replaced by another more tolerant
form. In areas of high pollution lichens may be found only on sites such as wounds on trees and
on sandstone walls, which have high (basic) pH. Scientists have found that, with considerable SO2
pollution in an area.

Lichens and Radiation

Lichens are also able to pick up and store radioactive fall-out, especially caesium and strontium.
The radioactivity harms particularly the cells which are part of the reproductive phase, such as the
spores of the fungi. Exposing x-ray film to radioactive lichens will leave spots or even the outline
of the lichen on the film, thereby revealing the history of their exposure to radioactivity. Some
hospital could make an x-ray film available for use. Place the lichen on top of a black x-ray package
and let it sit there for two weeks. Then develop the film. Although it may be seen utterly unlikely
that you would find any radioactivity in your lichens, some schools may find "positive" results.
After atomic testing both Alaskan Eskimos and Scandanavian Laplanders were found to have high
levels of radioactive contamination, which they had absorbed from eating reindeer, which in turn
ate lichens. The Chernobyl reactor accident brought similar dangers. Some classes might find it
an interesting exercise to sample with a geiger counter and begin to address some of the issues of
radioactive waste disposal. An inexpensive geiger counter available for classrooms is one made
by the Radalert Company of California. It runs about $225 as a kit and $300 assembled. Hand-
sized, it can be left counting, and gives total counts. Richard Borst's class in South Carolina did
indeed find a higher radiation count in the lichen taken from the Savannah River Plant.
Walking round the yards, park or other favorite natural environment, one could discover a
lichen. Lichen is very slow-growing, so they should not be disturbed but to examine to determine
if it is crustose, foliose or fruiticose. They can be identified by color: bright green, gray-green,
blue-green, yellow-green, or even pink! Generally speaking, the more lichen available or seen (in
color and quantity) the cleaner the air.
Analysis: Use the modified Hawskworth-Rose Index below to estimate air quality in the area.

1. No lichens present – very poor air quality

2. Crustose lichens only – poor air quality
3. Crustose and foliose lichens – moderate to good quality (based on number of different
lichens)
4. Fruticose, foliose and crustose lichens – very good air quality

Lichen as bio accumulator

Characteristics of bio accumulator according to Conti and Cecchetti (2000) and Stocker (1980)
and Philips (1977, 1980)

1. It accumulate the pollutant without however being killed by the level with which it comes
into contact
2. It must have a wide geographical distribution
3. It should be abundant, sedentary, or of scarce mobility as well as being representative of
the collection area
4. It should be available all year round and allow for the collection of sufficient tissues for
analysis
5. It should be easy to collect and resistant to laboratory conditions as well as being usable in
the laboratory studies of contaminant absorption if necessary
6. It should have high concentration factor for the contaminant under study and thus allow
direct analysis with no prior increase in concentration
7. It should have a simple correlation between the quantity of contaminant contained in the
organism and the average contaminant concentration in the surrounding environment
Lichen can be used as Index of Atmospheric purity (IAP). The formula with highest correlation
with pollution data is that which consider as a parameter only the frequency (F) of the lichen
species present in a sampling network comprising 10 areas.
IAP = ∑ Fi
F is the frequency (max. 10) of every ith species that is calculated as the number of rectangles
in the grid (a rectangle of the dimensions 30 x 50 cm each) in which a given specie appear.
BRYOPHYTES AS BIOINDICATORS AND MONITORING AGENTS OF POLLUTION
Bryophytes are green land plants which lack a vascular system and are simple both
morphologically and anatomically. The growth potential in bryophytes is not as highly polarized
as vascular plants. Bryophytes grow in a variety of habitats especially in moist places on soil,
rocks, trunks and branches of trees and fallen log. They obtain nutrients directly from substances
dissolved in ambient moisture. Some substances are probably absorbed directly from the substrate
by diffusion through the cells of the gametophyte. Bryophytes are used as reliable indicators of air
pollution. They are exploited as bryometers instrument for measuring hytotoxic air pollution. They
either independently or together with lichens can be valuable organisms in developing an index of
atmospheric purity (IAP) which is based on the number, frequency-coverage and resistance factor
of species. This index can provide a fair picture of the long-range effects of pollution in a given
area. There are two categories of bryophytes in response to pollution:
• which are very sensitive to pollution and show visible symptoms of injury even in the presence
of minute quantities of pollutants. This serve as good indicators of the degree of pollution and also
of the nature of pollutant
• which have the capacity to absorb and retain pollutants in quantities much higher than those
absorbed by other plant groups growing in the same habitat. These plants trap and prevent
recycling of such pollutants in the ecosystem for different periods of time. Analysis of such plants
gives a fair idea about the degree of metal pollution.

Effect of pollutants on bryophytes

There is an impoverishment of bryophytic communities in and around cities and industrial areas.
Urban areas comprise a series of habitats with a variety of substrates and moisture regimes and are
subject to varying levels of pollution. Bryophytes occupying certain substrates appear to more
sensitive to air pollution than others. Bryophytes have been disappearing from urban industrial
environments because of their sensitivity to polluted air. Species diversity in a polluted area varies
not only with the distance from the source of pollution but also with the type of substrate.

Air pollution inhibits gametangial formation and sexual reproduction in bryophytes. They also
reduce photosynthesis by degrading chlorophyll and growth of plants and eventually cause their
death. Metals and metalloid are accumulated by bryophytes from the substratum, wind-blown or
in wet deposition. When the metal enters the cell, it inhibits the photosynthetic activity. Enzymes
and membrane are poisoned when a heavy metal gains access to the cell interiors. Mercury is
particularly toxic low concentration greatly inhibited photosynthesis, temporarily increased
respiration, reduced chlorophyll levels and caused loss of intracellular K+ from Rhytidiadelphus
squarrosus. It is evidenced that when the pollution level goes down, the percentage frequency of
species goes up, which subsequently increases the fertility percentage. This situation varies from
species to species depending on the prevailing climatic conditions in the area. The pollutants can
affect organisms either individually or in combination of two or more. The common symptoms of
injury are plasmolysis and chlorophyll degradation in the leaf cells. SO2 exposed plants showed
brownish spots on the chloroplasts and plasmolysis in cells of leaves which contributed to the
ultimate death of the plants. When plants are exposed to SO2, it get absorbed in the plant tissue
which causes degradation of chlorophyll a by increasing the concentration of free H+ ions which
subsequently displace the Mg2+ ions from the chlorophyll molecule, converting it into phaeophytin
a. The typical response of mosses to SO2 pollution begin as a loss of colour at the tips of the more
exposed leaves and this gradually extends down the leaves and down the shoots until they have
lost all the chlorophyll. Chronic injuries such as growth retardation are seen. Low exposure of
plants to hydrogen fluoride (HF) results in the minimal injury and accumulation of fluoride to this
plant is low. Once hydrogen fluoride (HF) is absorbed on the plant surface, it moves towards the
tip of leaves causing a distinct pattern of injury which remain proportional to the exposure. Ozone
uptake by the plant species often results in acute injury, premature ageing and senescence.
Bryophytes show impairment of photosynthesis or increased membrane leakage when subjected
to an acute (150 p.p.b) ozone exposure. Several species of Sphagnum species were found to be
chronic to O3 exposure. Ammonia (NH3) is highly phytotoxic. Leaf tip chlorosis is followed by
necrosis when plants were exposed to NH3. Acid rain has two distinct effects like wet deposited
acidity and wet atmospheric deposition of the nutrients such as nitrogen and sulphur. Acidification
may damage cell membranes, solubilize potentially toxic metals like Al3+ and worsen the impact
of other pollutants like SO2. Acid rain produces drastic changes in the chemical properties of both
epiphytes and their bark substrates by reducing the pH of stem flow, increasing the proportion of
toxic bisulphate (HSO3 2-) ions in stem flow, decreasing the buffer capacity of the bark, decreasing
the internal pH of the epiphytes and increasing the metal concentration and chlorophyll loss in
epiphytes. Lead (Pb) pollution is caused by antiknock compounds consisting of tetramethyl lead
contents.
SEED PLANTS (GYMNOSPERM AND ANGIOSPERM)
INTRODUCTION
Seed plants are seed producing vascular plants. They are also called Spermatophyte. They have
well developed roots, stems and leaves. The seeds containing the embryo develops from a fertilize
egg of a very small gametophyte which is completely dependent on the sporophytes, the plant form
we see around us. The efficient seed dispersal of seed plants account for their continued existence
and widespread occurrence. The fertilization of the egg is by the male gamete which is brought by
pollination, followed by the growth of the pollen tube which carries the male gamete to the egg.
Water is not needed in this process, hence the seed plants are true land plants. The seed plants
includes the Gymnosperm and Angiosperms

General Characteristics of Seed Plants

They are terrestrially adapted i.e they are land plants. The gametophytes of seed plants become
more reduced and dependent on the sporophytes. They produce two types of spores (Megaspores
and Microspores) i.e. they are heterospores. They undergo pollution which replaces swimming as
the mechanism for delivering sperm to egg. They produce seed.

Gymnosperms
Classification
Gymnosperms are generally classified into four divisions. These are
- Coniferophyta - the conifers
- Cycadeophyta - Cycads
- Ginkophyta - ginko
- Gnetophyta - gnetae

Conifers
Among the gymnospems, the conifers are the most important. They have the following
characteristics: They are cone bearing plants with vascular tissue. All are woody plants; the great
majority been trees with just a few shrubs. Species can be found growing naturally in almost all
parts of the world. They are frequently dominating plants in their habitats. They are of immense
value, primarily for timber and paper production. The wood of conifers is known as softwood.
Examples are: Cedars, Cypresses, Pines, Redwoods etc.

External Morphology of Conifers

All are wood plants, and most are trees. Majority has a monopocal growth (ie. a single, straight
trunk with side branches) with optical dominance. Size varies from less than a metre, to over 100
meters. They are the world largest, tallest and oldest living things.

Ecological Adaptation of Conifer

The have distinctly scented pesin, which is secreted to protect the tree against insect infestation
and fungal infection of woods. They maintain high rates of photosynthesis at relatively low
temperature. Their needles (leaves) have thick warty coatings and sunken stomata which prevent
excessive loss. The sapwood column is large and acts as a short-term reservoir that supplies water
to foliage during drought periods.

A Confer
Gymnosperm

Angiosperms
The angiosperms are also known as flowering plants. They occupy every habitat on earth
except extreme environments. They live as epiphytes (i.e living on other plants); as floating
and rooted aquatics in both fresh water and Marine habitats, and as terrestrial plants that
vary tremendously in size, longevity and overall form. They can be small herbs, parasitic
plants, shrubs, vines or giant trees. They are also sources for other important resources such
as medicine and timber.

Classification
Angiosperm are generally classified into two (2). These are:
Monocotyledons (Monocots)
Dicotyledons (Dicots).

General features of Angiosperms

They could be photoautotroph, Saprobes or parasitic. Mostly pollinated by insects, birds, and other
animals, while some are by wind. They have vessels (xylem and phloem).
The monocots include lilies, grasses, corn, wheat, palms, while the dicots include roses, maples,
oaks, peas, and beans. Angiosperms possess certain external features that remarkably distinguish
them from other seed plants. This feature includes:

Flowers
They have modified leaves
Has sepals – usually green
Petals –brightly colored and insect pollinated
Petals –drab – wind pollinated
Has carpels – female reproductive organ.
Has stamen- male reproductive organ.

Fruits
It protects documents seeds.
They have mature ovary.
Simple fruit has single ovary flower (e.g. Pea pod, apple)
Aggregate fruit – produce from separate flowers (e.g pineapple)
Modifications for dispersal includes: attractive food, dispersal by wind, burns.

Estimation of relative water content (RWC)

RWC was determined according to Barrs and Weatherly (1962). RWC is a ratio of the amount of
water in the leaf tissue at sampling to that present when fully turgid. A composite sample of leaf
discs was taken and the fresh weight was taken and then leaf was floated on water for up to 24 h.
The turgid weight was then recorded, and the leaf tissue was subsequently oven-dried to a constant
weight at about 85C for 24 h. RWC is calculated by using the following formula:

(Fresh weight -dry weight) x 100

(Turgid weight- dry weight)
Indicator values for heavy metals- Plants take up many heavy metals, not only those which are
essential for a plant's metabolism such as cobalt, copper, iron, manganese, nickel and zinc, but also
elements which are not essential for plant growth such as Aluminium, cadmium, lead and thallium.
Many plant species have evolved ecotypes highly adapted to a surplus of one heavy metal or a
combination of several heavy metals (Ernst, 1974). This adaptation of a population to the
biologically available metal concentration of the soil is encoded only by a few genes specific for
each metal (Schat et al., 1996). This genetic design results in a high ecological specialization of
each ecotype with a strong impact on its performance on soils with another combination and
concentration of heavy metals, as shown for the Zn-Cd-ecotype from the metal-enriched soils near
Plombi6re (Ernst and Nelissen, 2000). Ellenberg and co-workers (1991) have given two values for
plant species which are pronounced (B) and moderately resistant (b) to heavy metals- the letter
[B,b] is derived from the German term "Blei ''= lead. In contrast to salinity metal-sensitive species
have not received an indicator value. Obviously the authors have recognized that they had to
differentiate between the differential demand and accumulation of all higher plants for cobalt,
copper, iron, manganese, nickel and zinc, and the different degrees of resistance to metabolically
non-essential elements such as cadmium and lead. Indicator values for heavy metals were
presented only for nine plant species with an "occurrence in habitats with high concentrations of
lead, zinc and other heavy metals. Some other species evolve resistant races but occur
predominantly on normal soils", but have not been considered in the evaluation.

Visible morphological and physiological changes as bioindicators of general metabolic

disturbance
Visible injury of plants are the result of insufficiem adaptation of a plant to changing
environmental conditions, either by natural processes such as the eruption of volcanoes, salt spray
or flooding, or by man-made processes, mostly caused by acute or chronic exposure to changes in
concentration of natural environmental components or to the release of new chemical compounds.
Visible plant damage by changes of leaf colour (chlorosis, necrosis) and changes of the growth
performance was first recognized by Stockhardt (1850) in the Harz mountains. He related negative
plant responses to high sulphur dioxide emissions.
Timely bioindication and biomonitoring have used these symptoms in evaluation of forest health
and of the disturbance of environmental processes.

Premature leaf losses

A severe impact on the metabolism of a plant will finally affect the longevity of plant or its organs.
A quite common reaction pattem to hazardeous environmental conditions is the premature loss of
plant leaves. Lack of needle classes in coniferous trees is a well-known example of the use of a
plant organ as bioindicator for the evaluation of the health of coniferous forests (Langeweg, 1988).
The factors causing leaf losses, however, may be quite different, ranging from exposure to air
pollutants, e.g. SO 2 and cement-kiln dust (Lerman and Darley, 1975), and increasing soil acidity,
to biota such as injury to ectomycorrhizal fungi, attack by parasitic fungi, e.g. Chrysomyxa (Crane
and Hiratsuka, 2000) and Lophodermium species on coniferous trees (Muller and Hallaksela,
1998), and by caterpillars, e.g. Thaumetopoea processiones on oaks, the latter also with strong
effects on human health (Hesler et al., 1999). Care, however, have to be taken that natural
processes are not confounded with anthropogenic impacts. The low number of annual year classes
on Scots pine in the Netherlands was first taken for the impact of acid rain, but part of the damage
was caused by Lophodermium pinastri on accessions from Poland and planted in the 1930s in the
country due to shortage of indigenous proveniances.

Malformation of plant organs

Plants which are insufficiently or not at all adapted to changing environmental conditions can
express disturbance of the metabolism by changes in their morphology, often visible as dwarfed
life forms (dwarfism, nanism) or gigantism of leaves. Growth performance of plants is very
responsive to a lot of environmental factors so that the reason for anormalities has to be stated by
(physico) chemical analysis, except if they are caused by animals, e.g. galls.

Malformation caused by radioactivity

Response to increased natural or industrial radioactivity (radiomorphosis, Savchenko, 1995) is
indicated by anomalies of plant growth or by irregular formation of leaves and flowers. On soils
naturally enriched by radionuclides (carnorite and monazite) in Brazil, India, Poland, Russia, West
African contries and the United States of America (Penna-Franka et al., 1965; Nair, 1961; Sarosiek
and Leonowicz-Babiakowa, 1970; Kovalskii et al., 1967; Osbom, 1961). Dwarfism of shoots and
misformation of flowers are frequent responses to enhanced doses of radionuclides. After the
Chernobyl disaster the emitted radionuclides have caused much morphological deviation in leaves
of oak and pine (Savchenko, 1995).

Malformation by a surplus of heavy metals

On soils over lead-enriched outcrops Papaver macrostomum shows misformation of the petals
(Maljuga et al., 1959) whereas dwarfism of plants and needles is frequently The use of higher
plants as bioindicators occurring in coniferous trees at exposure to increased levels of heavy
metals, such as Zn (Ernst, 1985) and Ni (Kozlov and Niemela, 1999). Deficiency of zinc is also
visible in dicots by stunted growth of the shoot, by shortening the intemodes ('rosetting') and by a
strong decrease in leaf size ('mottle leaves') (for a review: Baumeister and Ernst, 1978).
Asymmetry of Scots pine needles is another indicator of the impact of metal contamination
(Kozlov and Niemela, 1999)

Malformation by phosphorus deficiency

Phosphorus deficiency may be expressed by a reduction of leaf surface areas in crop plants
(Fredeen et al., 1989) and in wild plants. The needle length of Scots pine growing in the border
line of fens is often half of that of healthy ones due to phosphate fixation by iron. In contrast a high
degree of mycorrhizal colonization stimulates needle length (Timonen et al., 1997). All
malformations are good indicators of inadequate environmental conditions, but they demand a
(bio) chemical analysis to identify the kind and the quantity of the stressor.

Modification of tree-ring width and wood biomass

Radial growth responses of trees have received a lot of attention in reconstructing
palaeoclimatology (Fritts, 1976). It can also be used in the evaluation of the impact of other
environmental factors. It is long known that biological processes can diminish the annual
increment of ring width. An internal change of the allocation of carbon hydrates from wood to fruit
production in high fecundity (mast) years of beech and oak strongly affected ring width
(Rohmeder, 1967). A same effect can be caused by a mass development of defoliating insects
diminishing the photosynthetic leaf areas (Varley and Gradwell, 1962). Abiotic factors can have a
negative or a positive impact on wood diameter growth. A growing season with low precipitation
diminishes the annual increment (Fritts, 1976), but high precipitation stimulates it (Kozlowski,
1971). Similar contrasting responses can be evoked by high and low temperatures. The impact of
these natural sources of variation can be accentuated by anthropogenic factors: Diminished ring
width results from long-term exposure to a surplus of sulphur dioxide (Lux, 1965; Pollanschfitz,
1971; Grill et al., 1979) and heavy metals (Carlson and Bazzaz, 1977). Recently, the increase of
ring width in Pinus cerebra is related to enhance atmospheric CO2-concentration (Nicolussi et al.,
1995).

Table: Chlorosis and necrosis in leaves of higher plants caused by deficiency or toxicity of
chemical elements.
Chlorosis and necrosis
Disturbance of the chlorophyll synthesis often results in pale green to yellow leaves (lack of
chlorophyll synthesis, chlorosis) or brown leaves (breakdown of chloroplast pigments, necrosis).
Long-known is the lime chlorosis of plants suffering from a low iron supply on calcareous soils,
not only in crops (Bergmann, 1983; Chen and Hadar, 1991), but also in wild plants (Hutchinson,
1968). A deficiency of other nutritional elements, e.g. boron, magnesium, nitrogen, sulphur and
zinc causes also chlorosis (Marschner, 1995), often specifically expressed in leaves. If the amount
of heavy metals taken up by (even highly adapted) plants is surpassing the physiological
regulation, chlorosis will also appear (Ernst, 1999a). Recently chlorosis in Betula platyphylla var.
japonica is proposed as a bioindicator of soil acidification in Japan (Kitao et al., 2001). Changes
in the concentration of components of the ambient air can also cause chlorosis, as shown for Picea
0abies and Pinus sylvestris after exposure to enhanced concentration of volatile hydrocarbons
(Schr6der, 1998) or ozone (Utriainen and Holopainen, 1998). Selected sensitive cultivars, ecotypes
or varieties of plants may develop specific types of chlorosis (spickled, homogeneous, along leaf
veins) and necrosis (leaf margin, leaf tips). Spickled necrosis was caused by ozone in the tobacco
cultivar

Bioindicators of chronic exposure to changes in environmental compounds and processes

Now the main question arises: Can higher plants indicate chronic changes of environmental
compounds and processes and help to elucidate the reasons for the losses indicated by Red list
qualifications. The forest dye-off in many industrial areas and by long-distance transport of air
pollutants also in remote areas of the world [[are the result of long-term (chronic) exposure to
pollutants (cf. Smith, 1981). Some of the components of chronic exposure can be identified by a
thorough analysis of several of the affected plant species

Changes in air quality and radiation intensity

During the past 50 years human acitivities have changed the radiation intensity, especially in the
UV-B range (Rozema et al., 1997), the chemical composition of the atmosphere and the chemistry
of soils and waters by aerial fall-out. As a consequence, the genetics and physiology of organisms
and the composition of biological communities were modified by the exposure to these changes,
ranging in geographic scale from local to worldwide exposure. The greatest change in atmospheric
chemistry has taken place worldwide with regard to carbon dioxide. In Europe other pollutants
were formerly more important, such as sulphur dioxide and locally hydrogen sulphide. More
recently the air quality was modified by an increase in traffic-based emissions of nitrogen oxides
and by cattle-based emissions of ammonia

The response of plant species to the sulphur status of the environment. The negative effects of SO
2 emissions are the most investigated aerial pollutants from the 1850s onwards on coniferous trees
(St6ckhardt, 1850). When the concentrations of air pollutants pass a critical level, most plant
species suffer visible injury or disappear which is best documented for lichens. During the period
of high sulphur emission (1950-1968; Stuyfzand, 1993) many epiphytic lichens disappeared in the
industrial areas of Europe (cf. Wotterbeek et al., 2002) and after the reduction of the SO 2 emission
in Western Europe they are now strongly recovering, thus being good indicators of certain
components of air quality.

How have higher plants reacted to such an SO 2 exposure- Many plant species were highly
injured. The bleeching bark of Scots pine, the loss of needles, the above mentioned chlorosis and
necrosis of leaves of many plant species in the vicinity of SO 2 emission sources were good visible
symptoms of the affected metabolism (Mudd, 1975). But there was also another reaction pattern.
The increase of atmospheric SO 2 has consequently enhanced the deposition of SO 4 and increased
the sulphur concentration of soils. Because sulphur belongs to the major plant nutrients, positive
effects may be expected in higher plants

Exposure to enhanced levels of ammonia and nitrogen dioxide

Concentrations of NH 4 are increasing in agricultural areas. On a local and regional scale a lot of
ammonia is emitted into the atmosphere by the cattle-rearing industry in Belgium, Denmark, the
Netherlands and Northem Germany. Certain nitrophilous lichen species, e.g. Xanthoria parietina,
have shown a positive response to enhanced NH 4 concentrations (Van Herk, 1999). Higher plants
do not only respond to the nitrogen status of the soil (Marschner, 1995), but also to ammonia-
containing air. The latter can have serious impacts on individual plants, resulting in injured trees
of Pinus sylvestris (Van der Eerden, 1992), and on oligotrophic vegetation by stimulating the
growth of nitrogen-responsive species. The populations of the annual herb Ceratocapnos
(Corydalis) claviculata has strongly increased its occurrence and frequency in many forest types
in the vicinity of cattle-rearing areas in France, Germany and the Netherlands. Nowadays it is a
very common understorey species in broad-leaved and coniferous forests and a good bioindicator
of areas with high ammonia deposition (Schmidt, 1999; Decocq, 2000; Lethmate and Wendeler,
2000)

Element concentration for bioindication of changes in air quality -Only a few chemical
elements are directly taken up via the leaves i.e. C as CO, C02, CH4, volatile organic carbons
(VOCs) such as PCBs (Buckley 1982), F as HF (D~issler, 1976), N as NH 4, NO and NO 2
(Wellburn, 1990), O as O 2, 03 and peroxyacetylnitrate (Nouchi et al., 1984), and S as SO 2, H2S
and COS (Taylor et al., 1983). Many other chemical elements can be adsorbed to the leaf surface
and can be analysed in unwashed plant material. If there is a pollution gradient the element
concentration in the analysed plant part can help to identify and localize the emission source.
However, the co-occurring contamination of the soil by aerial fall-out, the uptake of the emitted
and deposited element by roots and translocation to the shoot will then be a combined indication
of air and soil contamination load at the site. The identified contamination gradient can vary from
a few metres as in plants and soils under copper high tension lines (Kraal and Ernst, 1976) up to
more than

Changes in soil quality- Diminishing the water table (desiccation), fertilization (eutrophication)
and the deposition of chemicals (acidification, contamination) will change the environmental
quality of a soil and consequently change the chemical composition of plants, the species
composition and diversity of the vegetation or the genotypes in a population.. Changes in chemical
composition of plants Whereas air pollutants will pass along the plant and one part of it will evoke
direct or indirect responses (Keller and Schwager, 1971), another part of air pollutants will be
deposited on the soil, thus increasing chronic exposure, and interact with the soil and its organisms
and afterwards with the higher plants. The high persistence of many chemical elements in the soils
can be analysed even if the original contamination source has ceased to cause new contamination.
After more than 5000 years smelting sites of heavy metals in the Bronze Age can still be identified
by enhanced metal concentrations in plants (Repp, 1963), often together with changes in vegetation
composition (Ernst and Nelissen, 2000). Prior to identify a contamination, a careful chemical
background analysis of the plant species under investigation is necessary due to the very species-
specific uptake and accumulation in leaves and other plant parts. Biological enrichment of an
element by its natural accumulation may be mistaken for environmental contamination. One of the
pitfalls is still the natural high concentration of manganese in Fagus sylvatica and the high zinc
concentration in Betula, Populus and Salix- species (Denaeyer-De Smet, 1970; Baumeister and
Ernst, 1978; Ernst, 1984)

Selection of ecotypes resistant to heavy metals Long-term exposure to soil contaminants has a
strong impact on the genetic composition of plant populations with many evidences for heavy
metal contamination. As mentioned above, wide-spread plant species are differentiated in local
and regional ecotypes with mostly a multitude of genotypes in the population. If the environmental
conditions are changing and the selection pressure is high, genotypes with often marginal presence
in the population can get their chance. Then selection in favour of the resistant ecotype takes place
within a couple of years or decades finally resulting in ecotypes with a high resistance to the
orginally adverse soil factor.
Selection of ecotypes resistance to pesticides -Frequent application of the same herbicide to
agricultural crops for a longer period let select within a decade herbicide-resistant weeds so that
the efficiency of the herbicide is lost. Although it was assumed for long time that this evolution
will not take place, there is now sufficient evidence that it has occurred and is still underway.
Herbicide-resistant populations evolved after long-term application of triazine herbicides in many
agricultural weeds, such as Amaranthus retroflexus, Capsella bursapastoris, Chenopodim album,
Poa annua, Senecio vulgaris, and Stellaria media (for a review: Warwick, 1991). Triazine-resistant
ecotypes of Senecio vulgaris had lower carbon assimilation and quantum yields than susceptible
ones, finally resulting in diminished growth and productivity (McCloskey and Holt, 1990). Due to
the high variability of these parameters in wild plant populations, a test of herbicide resistant can
only be done in the laboratory. Recently the frequent application of glyphosate has forced the
selection of glyphosate-resistant weeds, independently in Lolium rigidum in Australia (Powles et
al., 1998) and Eleusine indica in Malaysia (Lee and Ngim, 2000) despite the recent statement by
Baylis (2000) that it will not occur
Test plant procedure in the laboratory for hazard assessment
For establishing the toxicity of compounds prior to their release into the environment, several
toxicity tests have been developed for Regulatory Documents and Standard Test Procedures. The
general principle of all these tests was the cost efficiency often hampering or strongly diminishing
the biological quality, thus the relevance of the test.
The use of higher plants as bioindicators of the provenience, cultivar, variety or ecotype of the
species under consideration. Even then, the heterogeneity in a population will cause some minor
variation in the outcome of the tests. The endpoints of the test, however, will strongly depend on
the substrate. Artifical mixtures as those in the OECD (1984) procedure are not soils, but badly
defined components of clay and peat without describing the conditions for an equilibrium between
the test substance and the mixture, the pH of the mixture and the water holding capacity; there is
no standardization of the plant-specific growth conditions such as quality and quantity of radiation,
air humidity and air temperature, the addition of specific arbuscular mycorrhizal fungi and in the
case of legumes as test plants Rhizobium bacteria.

Germination as endpoint of the test- Due to economic arguments, the first life phase of a plant,
i.e. germination or seedling emergence is taken as endpoint of the test, not realizing that this life
phase is characterized by very population- and species-specific responses. Analysing germination,
will demand a very good definition of the endpoint. It should be the break-through of the radicula
through the testa which does mean that this observation cannot be made in a soil as substrate.
Another problem of the first life phase as endpoint is the impermeability of the seed coat to many
chemicals and the precipitation or complexation of chemicals in the cell walls. The quantification
of the endpoint is the percentage germination after a very short time, often no longer than 5 days
and a 50% decrease of seed germination is used for the ECs determination (USFDA, 1987;
USEPA, 1985). Many cultivars of agricultural crops have indeed a very rapid germination, but it
will be necessary that the maximum percentage of germination in the control has to be in the
vicinity of 95%. As soon as plant species from arable fields, grasslands and other ecosystems are
involved, the experiment has to be extended often for more than one year due to the genetically
and environmentally determined long-term dormancy (Baskin and Baskin, 1998) to achieve a
meaningful germination percentage. Germination of many plant species is governed by the
radiation quantity and quality, especially the ratio of red/far-red and temperature. Therefore the
procedure for germination test should clearly define all these abiotic test conditions.

Root elongation as endpoint of the test- As soon as the radicle is in contact with the environment,
nutrients and other chemical compounds can be taken up and affect the metabolism. It depends on
the seed reserve, how long the seedling will rely on its own sources and thus avoid the uptake of
extemal material. Root elongation is often one of the endpoints of the emergence tests because it
is long known that cell division and root elongation are often hampered at exposure to increased
concentration of a chemical. There are many procedures for the rooting test. The more recent
improvement is developed by Schat and Ten Bookum (1992) extending the experiment as long as
root elongation does stop (EC100) by dipping the root into a solution with active charcoal prior to
the transfer to a sequential increase of the concentration. One pitfall of the root elongation test is
the composition of the nutrient solution which has to be chosen as close as possible to the W.H.O.
Ernst natural nutrient supply. Growth of roots only in the solution of the chemical under
consideration will be disturbed by deficiency of major and minor nutrients and thus imbalancing
the integrity of the biomembranes. A second pitfall of the root elongation test is the root
morphology of a species. A plant species without a main (tap) root, but with a rapid development
of side-roots, stops soon elongation of the main root to enhance the development of side-roots for
a good exploration of the soil environments. With the exception of plant species belonging to the
family of Brassicaceae, Caryophyllaceae, Chenopodiaceae and Cyperaceae, the roots of most plant
species are shortly after emergence infested by mycorrhizal fungi. These fungi can modify the
toxicity of a substance; thus testing plant exposure to environmental compounds without addition
of the appropriate mycorrhizal fungus to the substrate will give a result which is not very relevant
for the response in a real environment (Joner et al., 2000). Although many root elongation
experiments are carried out with lettuce (without defining the cultivar), none of them reports the
involvement of a mycorrhizal fungus, thus the third pitfall.

Early seedling growth- The early seedling growth assay relies upon the development of the
seedling into the C-autotrophic stage (photosynthesis). In this case it is necessary that the seedling
is growing in either a nutrient solution well designed for the specific cultivar or in a soil which
chemistry and water supply is relevant for the cultivar and species under consideration. The
artificial mixture proposed in the OECD procedure (OECD, 1984) is not well defined (which type
of peat? which pH?) and environmentally not relevant. Seedling growth does not only rely on the
root environment, but depends strongly on radiation, air humidity, and temperature. So-called
"room temperature" is an ambiguous term ranging from 15~ in cool temperate laboratories up to
30~ in tropical laboratories. Many plant species demand a day/night cycle of radiation and
temperature for good growth performance. All these conditions have to be defined for a meaningful
test procedure, but are lacking in all procedures.

Life cycle bioassay- All plant species have to produce seeds to ensure the survival of the
population and the species. Therefore life cycle bioassays are the most biologically relevant
procedures in testing toxicity of environmental components because the plants passes all stages,
from the germination via the vegetative phase to reproduction. As shown for a Cd-Zn-resistant
ecotype Plombi6res of the non-mycorrhizal herb Silene vulgaris the response to metal-enriched
soils with different combination of Cd, Cu and Zn varied strongly between the various
developmental stages of the plants (Ernst and Nelissen, 2000). The life cycle of plants vary from
a month up to some hundreds of years. For economic reasons, life cycle bioassays are often
restricted to short living plant species or those which can reproduce already in the first year after
germination. There may be two endpoints of these assays, the total biomass and the amount of
seeds. Reproduction of many plant species demands the transfer of pollen either from neighbouring
flowers or from flowers of other individuals due to stigma incompatibility.