Term that describes a group of hundreds of chemicals

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What Is Dioxin?

Dioxin is the name generally given to a class of super-toxic chemicals, the chlorinated dioxins and furans, formed as a by-product of the manufacture, molding, or burning of organic chemicals and plastics that contain chlorine. It is the nastiest, most toxic man-made organic chemical; its toxicity is second only to radioactive waste. Dioxins and furans are some of the most toxic chemicals known to science.

Dioxin is a general term that describes a group of hundreds of chemicals that are highly persistent in the environment. The most toxic compound is 2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD. The toxicity of other dioxins and chemicals like PCBs that act like dioxin are measured in relation to TCDD. Dioxin is formed as an unintentional by-product of many industrial processes involving chlorine such as waste incineration, chemical and pesticide manufacturing and pulp and paper bleaching.

For women who eat moderate amounts of meat, fish, and/or dairy in industrialized countries, dioxin contamination of breast milk is 1-2 parts pertrillion dioxin.

Structure of Tetrachlorobenzodioxin

Dioxins form a family of toxic chlorinated organic compounds that bioaccumulate in humans and wildlife due to their fat solubility. The most notorious of those is 2,3,7,8- tetrachlorodibenzo-p-dioxin, often abbreviated as TCDD. The isomers containing chlorine in the 2, 3, 7, and/or 8 positions are the most toxic isomers and the ones which bioaccumulation.

Sources of Dioxin

Dioxins are produced in small concentrations when organic material is burned in the presence of chlorine, whether the chlorine is present as chloried ions or as organochlorine compounds, so they are widely produced in many contexts such as:

trash burn barrels

incinerators for municipal waste

iron ore sinter plants

incinerators for clinical waste

facilities of the non- ferrous metal industry.

Dioxins are also generated in reactions that do not involve burning â€" such as bleaching fibers for paper or textiles, and in the manufacture of chlorinated phenols, particularly when reaction temperature is not well controlled. Affected compounds include the wood preservative pentachlorophenol, and also herbicidessuch as 2,4-dichlorophenoxyacetic acid (or 2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T).

Health effects

Excessive exposure to dioxin may cause a severe form of persistent acne, known as chlorance.

Developmental abnormalities in the enamal of children's teeth.

Damage to the Immun system

Endometriosis

Birth defects

Diabetes

Incineration and Dioxin Emissions

Despite the potential as a long term hazard to the public health, waste treatment plants continue to be developed for the burning of dioxins into the atmosphere.

Does dioxin cause cancer?

The EPA report confirmed that dioxin is a cancer hazard to people. In 1997, the International Agency for Research on Cancer (IARC) part of the World Health Organization published their research into dioxin and furans and announced on February 14, 1997, that the most potent dioxin, 2,3,7,8-TCDD, is a now considered a Class1 carcinogen, meaning a "known human carcinogen".

What other health problems are linked to dioxin exposure

In addition to cancer, exposure to dioxin can also cause severe reproductive and developmental problems Dioxin is well known for its ability to damage the immune system and interfere with hormonal systems.

What are the health effects of dioxin on humans?

Studies have shown that dioxin exposure at high levels in exposed chemical workers leads to an increase in cancer. Other studies in highly exposed people show that dioxin exposure can lead to reproductive and developmental problems, increased heart disease and increased diabetes. Dioxins ability to cause birth defects (teratogenicity) has not been established in humans but studies in mice have shown that dioxin and similar chemicals can produce congenital defects.

In general the effects of dioxin on humans were only observed in populations that were highly exposed. The effect of the long term low level exposure that is normally experienced by the general population is not known. The long-term effects of dioxin exposure on human immunity, reproduction and development, and other organs and systems remain focal points for ongoing research, as are the molecular and cellular mechanisms by which dioxin causes these health effects.

What are the biological mechanisms of dioxin's toxicity?

The way in which dioxin affects cells is similar in some way to the way in which hormones such as estrogen work. Dioxin enters a cell and binds to a protein present in cells known as the Ah receptor. The receptor when bound to dioxin can then bind to DNA and alter the expression of some genes. This can lead to alterations in the level of specific proteins and enzymes in the cell. While it is not known exactly how changes in the levels of these different proteins cause the toxicity of dioxin, it is believed by most scientists that the initial binding of dioxin the Ah receptor is the first step. Ongoing Dioxin Research by the National Institute of Environmental Health Sciences. The health effects of dioxin on humans are the subject of ongoing studies at a number of research centers including the National Institute of Environmental Health Sciences in Research Triangle Park, N.C. In addition, NIEHS-supported researchers have studied the health effects of dioxin for more than 20 years and their efforts are reported in a large number of articles published in the scientific literature.

Dioxin and Cancer

The ability of dioxin to cause cancer (carcinogenicity) in laboratory animals is well established. The way in which dioxin cause cancer in animals and humans however is not fully understood, nor is it known if other dioxin-like compounds cause cancer. Humans are exposed to mixtures of dioxins and regulatory agencies such as the EPA regulate dioxin-like compounds together. Ongoing research at NIEHS is being conducted to examine the carcinogenicity of different dioxins, mixtures of these compounds, and the biological mechanisms responsible.

Dioxin and Agent Orange

Researchers are examining the effect of long term exposure to dioxin as a result of exposure to a Agent Orange, a defoliant used by U.S. forces in Vietnam. These studies have shown an elevation in diabetes in serviceman exposed to dioxin contaminated Agent Orange

Dioxin and Endometriosis

Studies have shown that dioxin exposure can increases the occurrence of endometriosis in monkeys exposed to low levels of dioxins. NIEHS grantees are extending these studies to examine the consequence of long-term exposure to dioxins on the incidence of endometriosis in women exposed to dioxin during a chemical accident in Seveso, Italy.

Dioxin and Immune function

One of the primary toxic effects of dioxin in laboratory animals is immune suppression, which results in decreased resistance to infectious agents and some cancers. The mechanisms and relationship between altered host resistance and immune dysfunction is complex, poorly defined, but extremely important to understanding health effects. NIEHS researchers are examining the mechanisms of immune suppression. Other studies are examining alterations in immune cell function in several human populations exposed to dioxin at both high levels and at low levels similar to that seen in the general US population.

Summary of Data Reported and Evaluation

Exposure data

Polychlorinated dibenzofurans (PCDFs) are formed as inadvertent by-products in the production and use of polychlorinated biphenyls (PCBs) and, in combination with polychlorinated dibenzo-para-dioxins (PCDDs), in the production of chlorophenols and have been detected as contaminants in these products. PCDFs and PCDDs also may be produced in thermal processes such as incineration and metal processing and in the bleaching of paper pulp with free chlorine. PCDFs are also found in residual waste from the production of vinyl chloride and the chloralkali process for chlorine production. The relative amounts of PCDF and PCDD congeners produced depend on the production or incineration process and vary widely. Like PCDDs, PCDFs are ubiquitous in soil, sediments and air. Excluding occupational or accidental exposures, most background human exposure to PCDFs occurs as a result of eating meat, milk, eggs, fish and related products, as PCDFs are persistent in the environment and accumulate in animal fat. High exposures have occurred in relation to incidents in Japan (yusho) and Taiwan (yucheng) involving contamination of rice oil and in accidents involving electrical equipment containing PCBs. Occupational exposures also may occur in metal production and recycling, and in the production and use of chlorophenols and PCBs. Based on limited data, the sum of the mean background levels of the penta- and hexachlorinated PCDF congeners commonly found in human tissues is generally in the range of 10-100 ng/kg fat, and the PCDF contribution to tissue international toxic equivalent (I-TEQ) values is typically of the same order of magnitude as that of the PCDDs. Since the mid-1980s, mean tissue levels of total PCDFs and PCDDs (measured as I-TEQ) in the general population have decreased by two- to three-fold. Five-fold higher tissue levels have been found in subpopulations consuming large amounts of PCDF-contaminated fish. Accidental exposures to PCDFs have led to tissue levels one or more orders of magnitude higher than background levels.

Origin of the Research Proposal

Dioxin is a general term that describes a group of hundreds of chemicals that are highly persistent in the environment. The most toxic compound is 2,3,7,8-tetrachlorodibenzo-p-dioxin or TCDD. The toxicity of other dioxins and chemicals like PCBs that act like dioxin are measured in relation to TCDD. Dioxin is formed as an unintentional by-product of many industrial processes involving chlorine such as waste incineration chemical and pesticide manufacturing and pulp and paper bleaching. Burning chlorine-based chemical compounds with hydrocarbons forms dioxin. The major source of dioxin in the environment comes from waste-burning incinerators of various sorts and also from backyard burn-barrels. Dioxin pollution is also affiliated with paper mills, which use chlorine bleaching in their process, and with the production of Polyvinyl chloride (PVC) plastics and with the production of certain chlorinated chemicals like many pesticide.

Polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) belong to the class of two-ring aromatic compounds containing one to eight chlorine molecules. The structures of PCDD/Fs are chemically very stable due to the absence of reactive groups, and hence these compounds are degraded very slowly in the environment (Pollitt, 1999). In addition to being recalcitrant in the environment, these compounds are highly toxic and carcinogenic (Pollitt 1999; Oh et al,.1999). The wide spread contamination of soil and sediment by PCDDs/PCDFs is currently the most visible environmental problem in many countries. The concentrction of dioxine in the soil is about 8000 pg TEG/g (Kazuei ishii et al 2004). Hence, these compounds are of great concern to the environment and human health. As biodegradation could provide an efficient, cost effective and environmentally benign means of remediation of these often-hazardous chemicals, this work is proposed for developing strategies for degradation of PCDDs/PCDFs compounds and thus for bioremediation of dioxins contaminated sites.

Aim and importance of this research in national international

This proposal is of great relevance to India, especially in view of the bioremediation of dioxin-contaminated sites. India is one of the main producer and consumer of many persistent organic pollutants (POPs) chemicals. In recent years, India has set many incinerators for treatment of municipal and medical wastes. Medical wastes incinerator is known to release high amount of dioxins. While various industrial practices have been attributed for the sources of PCDDs/ DFs in the environment, stringent regulations to control their emissions in several developed countries have decreased exposures and thereby concentrations in human sand wildlife in recent years (Liem et al 2000; Dyke et al 1997). Although studies have examined the occurrence of PCDDs/DFs in developed countries, little is known regarding the sources and exposure levels of humans and wildlife in India. Several chlorinated pesticides including 2,4-D and pentachlorophenol (PCP) are still being used in India (Gangal et al., 2000). Effluents that contain PCDDs/DFs from pulp and papers mills that use chlorine bleaching are discharged onto agricultural land for the irrigation of crops in certain parts of India (Kannan et al 1990). Similarly, chlor-alkali plants employing graphite electrodes are located throughout India (Anonymous 1995). Recently, presence of high levels of PCDDs/PCDFs and polychlorinated biphenyls in tissues of humans, fishes, chicken, lamb, goat, predatory birds, and Ganges River dolphins collected from various locations in India has been reported. Considering these, the contamination of soil by dioxins is highly probable. Therefore the bioremediation of dioxin-contaminated soils is essentially needed. Although dioxin degradation research well documented in many developed counties, there is not much work being done in India with regard to developing methods for the remediation of dioxins. Hence, the aim of this proposal is to carry out the dioxin degradation using environmental microorganisms.

Fate and toxicity of PCDDs/PCDFs compounds in the environment

Several of the PCDD/DF congeners, particularly those substituted at 2,3,7,8-positions, are persistent and highly toxic congener. The occurrence of PCDDs/ DFs in human tissues has been reported in the early 1970s and several studies have described the occurrence of these compounds in environmental media and biological tissues collected from various parts of the world (Giesy 1994). While various industrial practices have been attributed for the sources of PCDDs/ DFs in the environment, stringent regulations to control their emissions in several developed countries have decreased exposures and thereby concentrations in humans and wildlife in recent years. Dioxin toxicity posess a significant threat to human health, as it is found in the air, water, land, products, and food that we come into contact every day. Over 90% of the human intake of dioxins is through food, mainly from animal origin. The intake is ten to hundred times higher for breast fed babies than for adults with respect to their body weight. Dioxins are slowly bio-transformed in the body and are not easily eliminated. They tend to accumulate in fat and in the liver, by interacting with a cellular receptor, dioxins can trigger biological effects such as hormonal disturbances and alterations in cell functions. The mechanism of dioxin toxicity is similar in man and other vertebrates. Dioxin toxicity can cause cancer of the lungs, liver, kidney, breast, bone marrow, and tissues. Dioxin toxicity can also lead to reproductive, developmental, immune system, hormonal damage, birth defects, diabetes, skin conditions, excessive hair growth, endometriosis, learning disabilities, and a number of other health problems. The main PCDDs/PCDFs exposure from contaminated soil to human is skin contact. Thus biodegradation of dioxins is of great environmental concerns.

Microbial application of biodegradation

Degradative pathways of dioxins are widely distributed among bacteria. Dioxin degrading organisms can be easily enriched from the soil by using dinenzo-p-dioxin (DD) and dibenzofurans (DF) as the sole carbon source. Biodegradation of DD and DF by various bacterial strains has been studied. Previously, it was thought that due to recalcitrant nature of chlorinated dioxin compounds their use as carbon source by microorganisms are rare and if at all they are used biodegradation is never complete because of toxic chlorocatechol products. But recently, bacterial strains that are capable to degrading less chlorinated dioxins as their source of carbon has been discovered (Hong et al., 2004). Microbes degrading dioxins may contain enzymes yielding hydroxylated dioxins. Several key enzymes of the degradative pathway have been investigated in detail

The proposed objectives are selected based on the previous research work.

(III) Objectives

Extensive screening of soil samples collected from pesticide, paper industries, and incinerators for isolation and selection of fungi, which is capable of utilizing, chlorinated dibenzo-p-dioxins and chlorinated dibenzofurans as a sole source of carbon and energy.

Characterization of metabolites identified during dioxin degradation.

Production, purification, and characterization of the enzyme/enzymes involved in efficient dioxin degradation.

Identification of the involvement of genomic DNA or plasmid DNA in degradation of chlorinated dioxins.

(iv) Methodology

Microbial Isolates

Soil samples collected from different contaminated sites from industrial areas contaminated sites in Tamil Nadu will be screened for bacteria capable of degrading dibenzo-p-dioxins and dibenzo furans and chlorinated dioxins. The isolate showing efficient degradation of the above compounds will be selected for further study. The isolate will be identified and maintained as pure culture. Bacteria have the capacity to degrade a wide range of chlorinated dioxin congeners. Growth of the bacterium will be studied in the presence of Chlorinated dioxins, may serve as sole source of either carbon or energy or as co metabolites. Chlorinated dioxins degradation may either by angular or lateral dioxygenase enzymes. Utilization of Dioxins

Observing the change in maximum absorption for each compound will be screened for the efficiency of the bacterial isolate in utilizing chlorinated dioxins. The minimum inhibitory concentration (MIC) for each compound will be determined. The optimum conditions such as pH, temperature, nutrition, requirement of oxygen etc., for efficient degradation of chlorinated dioxins will be screened.

Characterization of metabolites identified during degradation of dioxins

The metabolites formed during degradation of dibenzo-p-dioxins and dibenzo furans and chlorinated dioxins will be extracted and identified with High Pressure Liquid Chromatography (HPLC), GC-MS spectra by comparing with authentic samples.

Identification involved in biodegradation of dioxins

This study is aimed at finding microorganisms capable of biodegrading dioxins efficiently to develop a biotechnological treatment for decomposition of dioxins. The enzyme involved in biodegradation dibenzo-p-dioxins, dibenzo furans and chlorinated dioxins will be studied. The enzyme will be assayed and enzymatic degradation of the above compounds will be carried out.

Studies on involvement of genes in degradation dioxins

The involvement of plasmid/genomic DNA in the degradation of dibenzo-p-dioxins, dibenzo furans and chlorinated dioxins will be identified. The gene responsible for the synthesis of enzyme involved in degradation of dioxins will be identified.

(V) Year-wise plan of work and targets to be achieve

I Year

1. Isolation and identification of dioxin degrading bacteria from different contaminated sites in Tamil Nadu.

2. Screening for the ability of bacteria to utilize dioxins and various chlorinated dioxins compounds as a sole carbon source.

3. Selection of bacterial isolate, capable of degrading chlorinated dioxins. Identifying and maintaining the culture in pure form and its morphological futures observed through Scanning Electron Microscopic (SEM).

4. Identification of the intermediates formed during degradation of dioxins and chlorinated dioxins using selected bacterium.

II Year

Identification of enzyme/enzymes involved in the degradation of dioxin and chlorinated dioxins.

Production, purification and characterization of the isolated enzymes.

Screening for involvement of plasmid/Genomic DNA in degradation of chlorinated dioxin.

Isolation and purification of Genomic/ Plasmid DNA from test isolates.

III Year

PCR primer selection and polymerase chain reaction.

Complete sequence analysis of the gene responsible for degradation of chlorinated dioxin.

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