Methods for Monitoring Environmental Pollution!
Environmental pollution poses a big threat to the healthy existence of humankind. The Governments world over pay serious attention to continuously monitor and minimize pollution.
The public and non-governmental organizations (NGOs) are also actively involved in this venture. Broadly, there are four levels of pollution monitoring agencies or environmental protection agencies (EPAs).
Primary level:
This is at the district or block level. The non-governmental organizations and the rural development agencies are involved in pollution monitoring.
Secondary level:
Existing at the state level, the monitoring is done by the respective state pollution control boards.
Tertiary level:
This is at the national/country level. Each country has its own environmental protection agency to monitor pollution.
Quaternary level:
International/inter- Governmental bodies are closely associated with monitoring of pollution which is a global phenomenon. World Health Organization and United Nation Environmental Programme are actively involved.
Biotechnological Methods for Measurement of Pollution:
In recent years, environmental pollution detection and monitoring is being done by approaches involving bio-systems. For this, purpose, several groups of plants, animals and microorganisms are utilized. The environmental protection agencies (EPAs) consider bio-monitoring of pollution as a useful device to monitor environmental pollution from the point of diagnostic, preventive and remedial measures.
Criteria for Bio-monitoring of Pollution:
The parameters or the criteria chosen for bio-monitoring of pollution are very crucial. They should be reliable, reproducible and cost-effective. Three types of criteria are mostly adopted for bio-monitoring of pollution-visual rating, genotoxicity rating and metabolic rating.
Visual rating:
In the visual rating, the growth rate and productivity are considered. When microorganisms are used in the test assay, the growth can be measured by turbidometric analysis. In case of higher plants, growth rate of different parts, visual damage to leaves, seed viability and germination frequency are taken into account.
As regards animals (fishes are commonly used), the concept of LD50 is used i.e. the dose at which 50% of the test organism is affected. Sometimes, the presence or absence of a particular species of an organism serves as an indicator for the environmental pollution.
Genotoxicity rating:
Genotoxicity tests measure the extent of damage caused to an organism by environmental pollution at the cellular and sub-cellular levels. The genotoxic lesions may be detected on the cellular organelles (membranes commonly used), genomes, immune systems, biomolecules, etc.
Cytotoxic tests such as measurement of chromosomal damage (including breakage), sister chromatid exchange (SCE) and micronuclei counting are also useful for pollution detection. The cell viability can be measured by detecting in vitro lysosomal viability. In recent years, DNA probes are in use for the identification of disease- causing organisms in water.
Metabolic rating:
The biochemical changes with environmental pollution can be measured (qualitatively and quantitatively) in selected organisms. In fact, certain metabolic parameters can be used as biomarkers to assess the pollution stress. The biomarkers used in metabolic rating include chlorophyll, proteins, nucleic acids (DNA and RNA) and changes in enzyme activities.
The biotechnological methods adopted for pollution measurement are briefly described in the following order:
1. General bioassays
2. Cell biological assays
3. Molecular biological assays
4. Biosensors.
Bioassays in Environmental Monitoring:
In the early years, the conventional physical and chemical methods were used for the detection of environmental pollution. Bioassays are preferred these days, since the biological responses that reflect the damages to the living organisms are very crucial for the actual assessment of pollution.
The organisms employed in the bioassays for pollution detection are expected to satisfy the following criteria:
i. It should readily take up the pollutant (absorption or adsorption).
ii. The organism should be sensitive to the pollutant.
iii. It should possess measurable features to detect pollution.
iv. The organism should have wide occurrence, and available round the year.
v. The bioassay should be simple, reproducible and cost-effective.
The most commonly used plants and animals in the bioassays are briefly described.
Plant test systems in bioassays:
Certain algae, bacteria, lichens, mosses and vascular macrophytes are commonly used in bioassays.
Algal bioassays:
Among the plant systems, algal bioassays are the most commonly used. Algae are considered to be reliable indicators of pollution due to their high sensitivity and easy availability, besides simple culturing techniques. The criteria adopted for algal bioassays are the growth rate, biomass accumulation and photosynthetic efficiency.
The algae used in the test assays include Chlorella, Microcystis, Spirulina, Navicula, Scenedesmus, Anabaena, Ulva, Codium, Fucus and Laminaria. In water, organic pollution can be detected by using the blue green algae, Microcystis, while metal pollution can be measured by Navicula.
Bacterial bioassays:
These are commonly used for the detection of fecal pollution in potable water, the most widely employed test being coliform test. Ames test that detects mutagenic pollutants is carried out by the bacterium Salmonella. Bacterial bioluminescence is a recent technique used for the measurement of gaseous pollutants and other compounds e.g. sulfur dioxide, formaldehyde, ethyl acetate. Photo bacterium phosphoreum is the organism of choice for bacterial bioluminescence.
Lichens in bioassays:
Lichens are widely used for the detection of atmospheric gas pollution, particularly in cities. Lichens are very sensitive for the measurement of sulfur dioxide.
Mosses in bioassays:
Environmental metal pollution can be detected by using certain forest and aquatic mosses e.g. Stereophyllum, Sphagnum, Brynus.
Vascular macrophytes in bioassays:
Water hyacinth (Eichormia crassipes) and duck weed (Lemna minor) are in use to detect aquatic metal pollution. In fact, certain biochemical parameters of macrophytes are used to serve as biomarkers of pollution e.g., peroxidase activity increases due to metal pollution; inhibition of nitrate reductase activity by mercury. The other commonly used bioassay parameters are the estimation of soluble proteins, nucleic acids, chlorophyll, and assay of enzyme (e.g. catalase, peroxidase) activities.
Pollution-induced peptides in bioassays:
Very recently, some workers have identified the presence of small peptides within the plant cells which are pollution-induced. These peptides, referred to as phytochelatins, are formed as a result of metal pollution. They are reasonably reliable for the detection of metal pollution.
Animal test systems in bioassays:
Among the animals, certain fishes, protozoa and helminthes are employed in bioassays.
Fishes in bioassays:
Toxic effects of environmental pollutants on fishes have been in use for quite some time as a measure of bioassays. In fact, the concept of LD50 (i.e. the dose of the pollutant at which 50% of the test organisms are affected) has originated from the studies on fishes.
The criteria or parameters used for assessment of fish bioassays include changes in the morphology and organs, behavioural pattern and modifications in metabolisms. The alterations in the enzyme acetylcholine esterase serve as a reliable marker for pesticide pollution. The most commonly used fishes in bioassays are Catla, Teleost, Labeo and Channa.
Protozoa in bioassays:
The ciliated protozoa serve as good bioassay systems for the detection of environmental pollution. The toxic effects of the pollutants can be measured by the changes in the behavioural patterns of protozoa, recorded on an ethogram.
Helminths in bioassays:
Rotifers are a group of helminths that grow on aquatic vegetation. They are used for the detection of organic matter in water (given by BOD). Rotifers, with round the year availability, easy cultivation, slow growth rate and easy recognition are used for bio-monitoring of water.
Pollution-induced peptides in bioassays:
As already described in case of plant bioassays (above), pollution-induced small peptides are found in animal cells also. They are collectively referred to as metallothioneins (comparable to phytochelatins in plants). Metallothioneins are useful for the detection of metal pollution.
Bio-monitoring of pollution with multiple species:
Most often, bioassays using a single organism are not adequate to detect pollution. In such a case, multiple species of organisms are used.
Cell Biology in Environmental Monitoring:
Cell biology deals with the study of the structural and functional aspects of cells and the cellular organelles. It is successfully exploited for environmental pollution detection, particularly with reference to mutagens and carcinogens.
The cell biological methods primarily aim to trace the harmful effects of pollutants on different cellular components — membranes, chloroplasts, mitochondria, chromosomes. In addition, the macromolecules namely nucleic acids (particularly DNA) and proteins are also used. Further, cell biological methods help in understanding the mechanisms of toxicity of pollutants.
Some important cell biological methods used in environmental pollution monitoring are described.
Membrane damage in bioassay:
The plasma membrane, an envelope surrounding the cell, protects the cell from hostile environment. It is the first cellular component to be directly exposed to pollutants. Many toxic substances that cause damage to cell structure and its functions are known. For the purpose of bioassay, the physical damages caused by pollutants or their deposition on the membranes can be detected by light, phase contrast and electron microscopy.
This approach may not be always practicable. The alterations in the semipermeable properties of the membranes due to pollutants can be detected by leakage of enzymes (e.g., lactate dehydrogenase), efflux of electrolytes or uptake of trypan blue. Lysosomes are also useful as biomarkers for measurement of cell viability. This can be done by neutral red retention test. The damaged lysosomes cannot retain this dye.
In recent years, animal and plant tissue culture techniques are also used for pollution monitoring. This is made possible by measuring cellular damages observed in cell cycles. A good example is the use of human lymphocyte culture to monitor the persons exposed to toxic pollutants.
Cytogenetic bioassays:
The genetic damages of the cells, as reflected by changes in the chromosomes, can be effectively used in bio-monitoring of pollution. For this purpose, animals (e.g. insect Drosophila) and plants (e.g. Arabidiopsis) with shorter life cycles are preferred. Other plants such as pea, maize and soy bean are also used in cytogenetic bioassays.
Chromosomal damage:
The pollutants may cause several types of chromosomal damages- fragmentation, bridge formation, and disruption in cell division. The chromosomal alterations can be effectively used for pollution detection. It has been clearly established that the severity of chromosomal damage depends on the chemical nature of the pollutant.
Micronucleus test:
Severe damage to chromosomes by pollutants may result in large scale fragmentation of chromosomes, followed by micronuclei formation. The degree of micronuclei development is directly related to the severity of the damage. Micronucleus test (MNT) is used for screening of mutagenic compounds.
Sister-chromatid exchange:
The damages caused by pollutants results in misexchange of chromosomal segments (chromatids) during cell division. The sister chromated exchange (SCE) can be detected by using a fluorescent dye technique.
Ames test in bioassays:
Ames test can be used for the detection of chemical mutagens and their carcinogenicity. This is very widely used bioassay for screening of various pollutants, drugs, cosmetics, food additives and metals. Ames test employs the use of a special mutant strain of bacterium namely Salmonella typhimurium (His–). This organism cannot synthesize histidine, hence the same should be supplied in the medium for its growth.
Addition of chemical carcinogens causes mutations (reverse mutation) restoring the ability of this bacterium to synthesize histidine (His+). By detecting the strain of Salmonella (His+) in the colony of agar plates, the chemical mutagens can be identified. The Ames assay can detect about 90% of the chemical carcinogens.
Recently, the yeast cells (Saccharomyces cerevisae) are also used for the detection of chemical carcinogens.
Molecular Biology in Environmental Monitoring:
The use of molecular probes and immunoassays in monitoring of environmental pollution is gaining importance in recent years. Molecular biological bioassays are particularly useful for the detection of bacteria, viruses and other pathogenic organisms that cause diseases.
DNA probes:
DNA probes and polymerase chain reaction (PCR) can be effectively used for water quality monitoring, particularly potable water. However, these techniques are expensive and not practicable at all places.
Immunoassays:
Immunological techniques are useful for the detection of pollutants (pesticides, herbicides) and identification of pathogens that exhibit immunological properties. Immunoassays are in use for the measurement of several pesticides e.g. aldrin, triazines DDT, glyphosate. Metabolic products of certain bacteria can also be detected by immunoassays. For instance, assay systems have been developed for the detection of toxins of cholera and Salmonella.
In recent years, use of monoclonal antibodies (MAbs) in the detection and bio-monitoring of environmental pollution is gaining importance. In fact, assay techniques are available for detection of pesticide and herbicide contamination in water.
Bioluminescent bioassays using Lux reporter genes:
Certain genes, referred to as Lux reporter genes, on the plasmids produce assayable signals. Whenever these genes are expressed in luminescent bacteria like Photo bacterium and Vibrio. Some bacterial strains have been developed through gene cloning (employing Lux reporter genes) for the detection of pollutants and their degradation. For instance, genetically altered Pseudomonas can be used for detecting naphthalene, xylene, toluene and salicylate.
Biosensors in Environmental Monitoring:
A biosensor is an analytical device containing an immobilized biological material (enzyme, organelle, cell) which can specifically interact with an analyte (a compound whose concentration is to be determined) and produce physical, chemical or electrical signals that can be measured. Biosensors are highly specific and accurate in their function. The details on biosensors—principles of working, types, various applications are described elsewhere Some of the important biosensors used in environmental pollution monitoring are briefly described.
BOD biosensor:
Biological oxygen demand (BOD5) is a widely used test for the detection of organic pollution. This test requires five days of incubation. A BOD biosensor using the yeast Trichosporon cutaneum with oxygen probe takes just 15 minutes for detecting organic pollution.
Gas biosensors:
Microbial biosensors for the detection of gases such as sulfur dioxide (SO2), methane and carbon dioxide have been developed. Thiobacillus-based biosensor can detect the pollutant SO2, while methane (CH4) can be detected by immobilized Methalomonas. For carbon dioxide monitoring, a particular strain of Pseudomonas is used.
Immunoassay biosensors:
Immunoelectrodes as biosensors are useful for the detection of low concentrations of pollutants. Pesticide specific antibodies can detect the presence of low concentrations of triazines, malathion and carbamates, by employing immunoassays.
Other biosensors:
Biosensors employing acetylcholine esterase (obtained from bovine RBC) can be used for the detection of organophosphorus compounds in water. In fact, portable pesticide monitors are commercially available in some developed countries. Biosensors for the detection of polychlorinated biphenyls (PCBs) and chlorinated hydrocarbons and certain other organic compounds have been developed.
Phenol oxidase enzyme (obtained from potatoes and mushrooms) containing biosensor is used for the detection of phenol. A graphite electrode with Cynobacterium and Synechococcus has been developed to measure the degree of electron transport inhibition during photosynthesis due to certain pollutants e.g. herbicides.
A selected list of environmental pollutants measured by employing biosensors is given in Table 54.1.
