33 Bioremediation of Pesticides

Dr Abhijeet Joshi

1.Introduction

2.Nature of Pesticides

3.Bioremediation Strategies 

3.1. In situ bioremediation

3.2. Ex situ bioremediation

3.3. Microbial degradation of pesticides

3.4 Enzymatic Degradation of Pesticide 

3.5. Pesticide Conjugation

3.6 Bioremediation using Fungi

3.7 Phytoremediation

3.8 Limitations of Bioremediation

1.Introduction

Pesticides are chemical compounds that are widely used in the agricultural sector for killing pests that infest crops and thereby increase the yield of crops. Due to high demand of food to support overgrowing population farmers are inclined towards excessive use of pesticides. Pesticide usage overall has reached about 2 million Tonnes in the world. In India also the use of pesticides occur at 0.5 kg/hectare which makes India among the largest producer of pesticides. In India, consumption of insecticides is more in comparison to herbicides which are more used in other countries. This may be due to warm humid climatic conditions. Only about 2-3 % of these pesticides are used and remainder of pesticides remain in soil and water (1). This has resulted in contamination of agricultural fields, infiltration of pesticides into the food items and consequently entry into human body. In India Endosulfan, Chlorpyrifos, Atrazine, Carbaryl, Cypermethrin, and Carbofuran are some of the commonly used pesticides. Consumption of pesticides has led to deleterious effects on human health.

World Health Organization classifies pesticides based on the hazard it may cause, like extremely hazardous (Class 1A), highly hazardous (Class 1B), moderately hazardous (Class 2), and slightly hazardous (Class 3). Hypersensitivity, immunological reactions like eczema, dermatitis, respiratory diseases and infections, neuropathy, toxicity to liver, kidneys reproductive organs, Parkinson’s and Alzheimer’s diseases. Pesticides can cause mutations in genetic material in humans which can lead to carcinomas. Environmentalists around the world are working towards solving this problem. Transport and behaviour of pesticides is completely dependent on chemical nature, solubility of pesticides, adsorption in soil, input, removal, degradation by physical and chemical means etc. The traditional methods for remediation of pesticides like incineration using high temperature, landfilling and chemical degradation have certain drawbacks like complexity, high cost and the danger of being exposed to contaminants for the workers working at the site as well as nearby residents by causing air pollution.

2.Nature of Pesticides

Pesticides are widely used xenobiotic natural or synthetic chemicals. Mostly the pesticides resist their biodegradation. They can be classified based on organisms, based on mechanism of toxicity, rote of administration or based on chemical nature of pesticides as described in Figure 1. Among all the classification systems chemical classification is the most common and useful way (2). Chemically pesticides comprise of organochlorines, organo-phosphorous, carbamates and pyrethroids. Organochlorines have many chlorine atoms in their chemical structure and were among initial synthetic organic pesticides in agricultural and health sector. Organochlorines cause disruption in nervous system leading to convulsions and paralysis. Commonly used organo-chlorine pesticides include DDT, Aldrin, chlordane, endosulfan etc.

Figure 1: Different classification of pesticides commonly used in agricultural sector

Organophosphates are compounds having phosphate functional groups in their formula and are found to be more toxic as it acts on enzyme cholinesterase which is involved in degradation of acetyl choline neurotransmitter. The toxicity causes permanent action of acetyl choline across synapse causing paralysis and death. Organophosphates are found to be non-persisting in the environment as they can be easily degraded. Some commonly used organophosphates include parathion, diaznion, malathion and glyphosate. Carbamate pesticides are derivatives of carbamic acid and also function as cholinesterase inhibitors. Choline esterase inhibition due to carbamates is found to be specific and reversible. Some common pesticides belonging to this class include carbaryl, carbofuran, and aminocarb. Pyrethroids are naturally derived compounds obtained from plants of pyrethrum (Chrysanthemum cinerariaefolium). Pyrethroid insecticides are fast degrading by

photochemical modes and cause knocking down effect in insects with low toxicity in mammals. Some common examples of pyrethroid insecticides include permethrin, deltamethrin and cypermethrin. Recently biopesticides have been also developed which are derived from natural materials. Environmental persistence of pesticides can vary to a great extent for example Organochlorines (2-5 years), Organophosphates (1-12 weeks) and Herbicides (4-50 weeks). Degradation and mineralization are common terms employed for describing bioremediation (3). Physico-chemical properties and environmental factors affect the pesticides chemodynamics. Degradation of pesticides can lead to generation of toxic products or non-toxic compounds. Chemical degradation of pesticides may constitute processes like reduction, oxidation, hydrolysis and photolysis whereas biological degradation occurs due to similar processes occurring due to microorganisms. Due to the hydrophobic nature the pesticides get adsorbed to soil and are entrapped in the pores of soil.

The entrapment of pesticides in the soil makes them less available for degradation by any mechanism. In most cases the degradation of pesticides result in detoxification, however in some cases like metabolism of Propanil which is used as an herbicide are degraded to tetrachloroazobenzene compounds which are carcinogenic in nature and may lead to severe health concerns. Lipophilic pesticides are also prone to a process called biomagnification where in persistent molecules concentration accumulates in the cells and can increase up-to three times. At higher trophic levels of food chain the concentrations of pesticides go on increasing. Due to this magnification effect the pesticides can be highly toxic and can cause death or serious implications in health of organisms.

3.Bioremediation Strategies

Biodegradation involves breakdown of pesticides forming water and carbon dioxide and oxides of mineral salts. Metabolism of pesticides into inorganic compounds is called biomineralization. Microbes present at the polluted sites continuously detoxify the pesticides present in the ecosystem. However the process may not be enough to completely remove or treat high concentrations pesticides from a given site. The methods of remediation should be ideally safe and the intermediates produced should be non-toxic. Success of a bioremediation technique requires availability of pesticide to microbes, environmental conditions

3.1. In situ bioremediation

These techniques allow the treatment of pesticides in place, thus reducing excavation as well as transport of contaminants. In situ bioremediation is less expensive and can be conducted through several different medium, such as using fungi. White rot fungi contains lignin which is a recalcitrant compound and this fungus contains enzymes known to degrade lignin, and other toxic as well as recalcitrant compounds.

a) Bioventing: This technique involves the injection as well as even distribution of oxygen and/or nutrients such as nitrogen and phosphorous into the soil for bioremediation (4). Actually that distribution depends upon soil texture. Bioventing works well for properly drained, medium as well as coarse-textured soils. A basic bioventing system employs the use of a well and a blower that pumps air into the soil passing via well.

b) Bio-sparging: This technique involves the injection of air under pressure below the water level so that concentrations of oxygen in the water table increase which subsequently improve the degradation potential of naturally occurring bacteria (5). This enhances the mixing in the saturated zone and thus increases the contact between groundwater and soil.

c) Bio-augmentation: Importing microorganisms to a polluted site to increase rate of decomposition is called as bioaugmentation (6). This technique is generally combined with biostimulation which involves sufficient quantity of water, oxygen, and nutrients etc. that are instilled in the sites with pollution so that the activity of microbial degrading population is increased through the accumulation of nutrients, amendments and other limiting factors.

The following are the limitations of in situ bioremediation: 1) it does not work for all kinds of soils, 2) it’s difficult to attain complete degradation and 3) It’s difficult to optimise natural conditions such as temperature for proper biodegradation. Bioventing and Biosparging are not suitable for compounds which get volatilized very quickly. Bioventing and Biosparging have not been in use for degradation of pesticides.

3.2. Ex situ bioremediation

Ex situ bioremediation involves the measures to treat the polluted soil at a site different from its original location (4). Following are different treatments used:

a) Land-farming: In this process the soil is excavated and separated by sieving. The contaminants present in the soil are degraded and immobilised by placing the polluted soil in layers over clean soil. The contaminated soil layer is then covered with a clay membrane and the next step involves the addition of oxygen by ploughing or milling. The pH of the soil is maintained using crushed limestone. The mixing of contaminated soil with clean soil allows for microorganisms to interact with pollutants and degrade them. Land farming is the most widely used bioremediation technique (7). Organophosphate pesticides like malathion, parathion and metolachlor and traizine can best be degraded using aerobic methods of land farming.

b)Biopiling: The biopile system consists of a bed of contaminated soil for treatment i.e. mounds of contaminated soil an aeration system, an irrigation/nutrient system and a leachate collection system. Moisture, nutrients, heat, oxygen and pH are the factors that are controlled to increase biodegradation. The nutrient system is buried under the soil to pass air and nutrients either by vacuum or positive pressure. Treatment time is usually for 3 to 6 months. The bio pile can be made into anaerobic environment using covered plastic sheets so that the aerobic organisms utilize the oxygen in the soil. In oxygen-less environment the anaerobic microbes will proliferate which have capability to degrade organic pollutants like pesticides.

c) Composting: In this process microorganisms degrade organic wastes at temperatures usually in the range of 55° to 65°C which increases the solubility of contaminants and results in higher metabolic activity in composts (8). The contaminated soils are first excavated as well as screened to remove large rocks and debris. The soil is then taken to a composting pad and provided with protection from weather extremes. Manure, agricultural wastes as well as wood chips are used as a supplemental carbon source. Soil and amendments are layered into long piles known as windrows. After the completion of the entire composting period, the windrows are disassembled and the compost is taken to the final disposal area after disassembling the windrows. Composting can also be done as an in situ method, however it is not a preferred method (8).

d) Precipitation or flocculation: This method is being highly considered for remediating ground water containing heavy metals as well as their radioactive isotopes. This method involves converting a soluble heavy metal salts to insoluble salts that precipitates. The precipitate is then removed from the treated water by physical methods such as clarification (settling) and/or filtration.

3.3. Microbial degradation of pesticides

Bacteria species that degrade the pesticides belongs to genera Flavobacterium, Arthobacter, Aztobacter, Burkholderia, and pseudomonas. Pesticide biodegradation involves oxidation of chemical compounds and formation of carbon dioxide and water with the release of energy. The released energy and the pesticides act as energy source for the microbes which is utilized for their growth (9). In case the innate microbial flora is not able to degrade the pesticides externally added microflora will be useful for improved degradation. The added

microorganisms can be either occurring naturally or genetically engineered. The addition of microorganisms to contaminated environments may not be successful to a great extent as they may not be able to survive in the new environment period for a long time. An alternative to this strategy is to introduce specific genetic modifications to in bacteria so that they adapt well and are able to degrade the pesticides efficiently. The genetic modification can be done by introducing genes directly in the environment and natural uptake and transformation helps the survival of microorganisms or genes via conjugation in microorganisms. The degradation of pesticides is dependent on availability of pesticides to organisms, state of microorganisms, proliferation of micro-organisms and number of microorganisms. In some experiments cow dung has been used for bioremediation. The cow dung slurry contains huge microbial biomass. Nutrients along with this microbial population in cow dung slurry pesticides have been found to be detoxified.

Degradation of pesticides by microbes also depends on environmental conditions like pH, temperature, and nutrients (10). Pesticides can be degrading easily or recalcitrant due to presence of negatively charged species in the molecule. Besides organ phosphorus compounds, the neonicotionoids are degraded by the Pseudomonas species. Bacterial degradation is also controlled by presence of anionic species like chloride, sulphate etc. which are attached to hydrocarbon ring. These anions prevent microbes attacking ring structure. Lesser bioavailability of pesticides is an important challenge to be dealt with for success of bioremediation. Bio surfactants can aid in improving the bioavailability which increase the uptake of pesticides in the cells (11). In addition, use of metal particles can serve as reductant and catalysts for environmental contaminants.

3.4 Enzymatic Degradation of Pesticides

Enzymes can degrade pesticides through intrinsic detoxification mechanisms, metabolic resistance, and via biodegradation by soil and water microorganisms. Fungal enzymes like laccase, oxidoreductases and peroxidases have significant application in removal of pesticide contamination in fresh, marine water or terrestrial (12). The enzymes have a significant part in the biodegradation of pesticides. The enzyme can be produced internally or externally from the cells of microorganism. The absence or low concentration of pesticide degrading enzyme is the main reason for persistence of pesticides in the environment. Oxygenation is a key step in pesticide degradation which can be catalysed by oxidative enzymes. Cytochrome P450 is one of the most commonly employed oxidative enzyme in pesticides degradation which results in hydroxylation of substrates (13). Enzymes like peroxidase, polyphenol oxidase, tyrosinase, laccase etc. also catalyse oxidative reactions. Hydrolytic enzymes like esterase, lipases, proteases help in cleaving bonds and adding -H and -OH to the molecule. They are more effective in compounds like amide, ester and carbamate functional groups. Pseudomonas fluorescens has been characterized with different types of esterases each present in different organelles and with different protein structure. Nitro-aromatic pesticides can be metabolized by nitro-reductase enzymes which are commonly found in aerobic and anaerobically metabolizing bacteria. Organophosphates are degraded by bacteria like E. Coli which makes these pesticides degradable due to enzymes like C-P lyase.

3.5. Pesticide Conjugation

Pesticide conjugation is process in which externally added/internally present compounds are linked to pesticides or their degradation products which in turn help in their detoxification. Existing enzymatic systems are used in this process for example Uridine diphosphate-glucosyl (UDPG) transferase is involved in pesticide-glucose linkages and pesticides-glucose esters (14). Typical reactions involved in pesticide conjugation reactions include xylosylation, acylation, alkylation, nitrosation etc. Methylation is a common reaction utilized by bacteria and fungi for detoxification of pesticides (12). Example of such a reaction is degradation of a herbicide metbromuron by microbes to 4- bromo aniline which is then acylated to from 4- bromoacetanilide. Pesticides are often found to be linked with cell walls of plants. The relevance of these bound pesticides for well-being or toxicity is not studied till now.

3.6 Bioremediation using Fungi

Bioremediation based on fungi is also called Mycoremediation. Fungi are responsible for certain minor structural changes in the pesticides to degrade them into nontoxic compounds and liberate them into soil where it can be degraded further (15). Many fungi like Aurcularia auricular, Agrocybe semorbicularis, Dichomitus squalens, Flammulina velupites, Avatha discolor, Stereum hirsutum, Pleurotus ostreatus, Hypholoma fasciculare etc.). White rot fungi, from the family Phanerochaete, are recognized for efficient degradation of toxic chemicals (15). White rot fungi is also known for bioremediation of organochlorines. They can also be used for inoculation during the composting process. Some of the fungi produce extracellular enzymes that have properties to degrade organic compounds in presence of hydrogen peroxide. Hydrogen peroxide is produced by these fungi and making them useful for organic decontamination. Fungi have been found to degrade chlordane, Lindane and DDT (16). However, huge concentrations of fungi would be required for bioremediation of polluted sites due to their slow rate of degradation.

3.7 Phytoremediation

Use of plants to remove pollution is called as phytoremediation. It is gaining recognition because of its in expensive and aesthetic remediation method. Several compounds derived from plants are used as biopesticides. Some compounds like polyamines enable plants to sustain with pollutant concentrations which are very high and allow for increased uptake of pollutants. Phytoremediation is a simpler technique of solving environmental pollution in comparison to microbial bioremediation (17, 18). Some examples of plants which are genetically altered to increase the uptake and metabolism include A. thaliana, tomato, tobacco and soyabean where in enzymes like cytochrome P450 and glyphosate oxidase are expressed. Herbicides are meant for killing plants hence such pesticides are difficult to be detoxified using phytoremediation. Phytoremediation may involve phyto-degradation which metabolism of organic pollutants using plants and phyto-volatilization which is evaporation of organic pollutants as they pass through the leaves of plants. This allows for exchange of pollution from one mode to other. Contaminants can also get immobilized in the plant structures in a process called as phyto-stabilization. Phyto-stabilization can occur in any kind of plant vegetation and will prevent soil contamination using organic compounds. In the soil root interface, rhizospheres are present which have a good population of microbes. Due to high microbial metabolic activity rate of biodegradation of pesticides is higher. In phyto- stimulation the plants are not directly involved in bioremediation, however they act as medium for promoting the microbial growth which in turn act as bioremediation agents. Major limitation of phytoremediation is that plants grow only in top three feet of soil but the pollution of pesticides may be occurring deeper than 3 feet. Another fact is that if the contaminants are not metabolized by plants then they can get accumulated in different parts of plants. This accumulation might cause toxicity to herbivorous animals.

3.8 Limitations of Bioremediation

Major limitation is the type of organisms, their nature and capability to metabolize pesticides. To stimulate the pesticide degradation environmental conditions need to be optimized extensively so that the degradation rate can be enhanced. Microorganisms need to be exposed to low concentrations of pesticides so that they develop mechanisms to degrade the pesticides. Sometimes fertilizers and/or aeration are required for proper nutrition of microbes. The addition of fertilizers and oxygen may affect growth of microbes which are natively present especially in cases of in situ bioremediation. Further the cost involved in bioremediation process is huge and the benefits obtained are very less which has not favoured any industrial input in this fields. The measure of damage done by the bioremediation process to the environment in terms of altering environmental conditions, introduction of non-native species of microorganisms have not been quantified in both ex situ and in situ methods.