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JUNE, 2012

CERTIFICATION I certify that this research work was carried out by Njoeteni Kate Agatha in the Department of Environmental Science Technology (ISLT) Botany Department, Delta State University, Abraka under my supervision.

_________________________ NJOETENI KATE AGATHA (PROJECT STUDENT)

_______________ DATE


_______________ DATE

_______________________ DR. (MRS) EDEMA (HEAD OF DEPARTMENT)

________________ DATE

DEDICATION I dedicate this project work to God Almighty for his love, mercies, grace, encouragement and protection upon my life through out my study in Delta State University, Abraka.

ACKNOWLEDGEMENT I am most grateful to God Almighty for his Grace and Wisdom in seeing me through School and for making my project work so easy. I wish to express my appreciation to my loving parent Mr. and Mrs. Njoeteni for their empowerment, advice and care. Indeed I am forever grateful to them. I appreciate my project supervisor, Mr. Ehwarieme D. Ayo, for his cooperation through out. Also my gratitude goes to all lectures of the Delta State University, Abraka. I also say thank you to all my fellow project group students for their togetherness through out the period of the project work. To the entire Environmental science students admitted in the 2006/2007 academic section, I say I love you all. Thank you also goes to Mr. Obiora, head of laboratory unit of the Department of Petroleum Resource (DPR) Warri, Delta State for his fatherly advice and attention.


Title page

i ii iii iv v vi

Certification Dedication -


Acknowledgement Abstract -


Table of content

Chapter one 1.0 1.1 IntroductionWhat is DDT - - - 1 2 2 3 4 4 6 8 9 9 10 10 11

1.1.1 Origin and history of DDT 1.1.2 Chemistry of DDT 1.2 Application of DDT -

1.2.1 Application of DDT to the Environment 1.2.2 Application of DDT as an insecticide 1.2.3 Application of DDT for disease control1.3 Effect of DDT 5

1.3.1 Effect due to Transport of DDT 1.3.2 Effect due to Metabolism of DDT 1.3.3 DDT effect on health -

1.3.4 DDT Effects on children -

1.3.5 DDT effect to non laboratory mammal1.3.6 DDT effect to microorganism -

13 13 13 14 15 19 20 20 21 25 25 26 26 26

1.3.7 DDT effect to aquatic invertebrates 1.3.8 DDT effect on egg shell- -

1.4 Fate of DDT to the Environment 1.5 Biodegradation of DDT -

1.5.1 Biodegradation of DDT in air

1.5.2 Biodegradation of DDT in water -

1.5.3 Biodegradation of DDT in sediments and soil - - 1.6 Current status of DDT - - - - - - - 1.7 Aims and Objective1.8 Research problems1.9 Scope of the study1.10 Limitation of studyChapter two 2.0 Collection of soil sample - - -


2.1 Analysis on the physical and chemical properties of the samples- -28 2.1.1 Particle size analysis 2.1.2 Chloride determination 28 28 28 29

2.1.3 Determination of exchangeable cations 2.1.4 PH determination 2.2 -

Isolation and Enumeration of bacterial in the soil



29 29

2.3 Sub-culturing 2.4

Isolation and enrichment of DDT degrading bacterial Isolates 30

2.5 Determination of growth profile in different concentration Of DDT31

2.6 Shake-flask biodegradation of the DDT by the bacterial Isolates 31

2.7 Identification and characterization of the isolated Bacteria 2.8 Gram staining2.9 Biochemical test Chapter three 3.0 Results Chapter Four 4.0 Discussion 4.1 Conclusion Appendix References 46 49 50 53 36 32 32 33


Dichlorodiphenyltrichloroethane (DDT) is an organochlorine. It is a highly hydrophobic, colourless, crystalline solid with a weak chemical odour. DDT is very effective in killing mosquitoes, but when used, is persistent in the environment, and has the ability to bioconcentrate in the food chain. One way to remove the impact of DDT in the environment is biodegradation. The soil sample worked on was collected from uncultivated site of the Delta State University, campus 3, Abraka, Delta State. Ten samples were collected and worked on, having bacteria count of 1.01105 -1.09107 cfu/mL. The six bacteria isolated were Bacillus sp., Micrococcus sp., Proteus sp., Pseudomonas sp., Enterobacter sp. and Staphylococcus sp. A minimal salt medium was used for the enrichment and degradation potential of DDT. Bacteria isolated were inoculated into different concentrations of DDT minimal salt medium (5ppm, 10ppm, 15ppm, 20ppm and control), to determine the tolerance level and degradation of DDT as a sole carbon source. The bacteria able to degrade DDT were Bacillus sp., Micrococcus sp., and Pseudomonas sp. Optical density reading was observed to increase steadily until about the 20th day, before dropping gradually. Also the pH reading reduced from 7 to 4.5 from day 0 to 25.The role of microorganism in the degradation of pollutants like DDT has long been recognized. Areas which are contaminated with DDT can be remediated with these bacterium agents, which are safe and have the ability to degrade DDT, there by reducing its polluting level to the barest minimum in our environment.


GENERAL INTRODUCTION Dichlorodiphenyltrichloroethane (DDT) is still one of the first and most commonly used insecticides for indoor residual spraying because of its low cost, high effectiveness, persistence and relative safety to humans (Hecht et al., 2004). It is therefore a viable insecticide in indoor residual spraying owing to its effectiveness in well supervised spray operation and high excite-repellency factor. Although DDT is very effective in killing or repelling mosquitoes, its use has been severely reduced and restricted to indoor residual spraying, due to its persistence in the environment and ability to bio concentrate in the food chain (Cousins et al., 1998), (Hickey, 1999). One of the removal processes with significant impact on the fate of DDT in the environment is biodegradation (You et al., 1995). Biodegradation and bioremediation are matching processes to an extent that both of these are based on the conversion or metabolism of pesticides by microorganisms (Hong et al., 2007). A successful bioremediation technique requires an efficient microbial strain that can degrade largest pollutant to minimum level (Kumar, et al., 2006). The rate of biodegradation in soil depends on four variables: 1. Availability of pesticide or metabolite to the microorganisms 2. Physiological status of the microorganisms 3. Survival and
4. Proliferation of pesticide degrading microorganisms at contaminated site

and Sustainable population of the microorganisms (Dileep, 2008). Therefore, to attain an achievable bioremediation, it requires the creation of unique niche or microhabitats for desired microbes, so they can be successfully exploited. So far, no micro-organisms have been isolated with the ability to degrade DDT as a sole carbon and energy source (Jacques et al., 2008), but organisms may degrade the organochlorine via co-metabolism under aerobic or anaerobic conditions. Most reports indicate that DDT is reductively

dechlorinated to DDD under reducing conditions (Lai et al., 1999). Extensive biodegradation of DDT and DDT metabolites in some bacteria has been demonstrated (Aislabie et al., 1998). The major bacterial pathway appears to involve an initial reductive dechlorination of the trichloromethyl group to form DDD. Further dechlorination to other intermediaries occurs resulting finally into non chlorinated compounds which are not harmful to the environment. 1.1 WHAT IS DDT: DDT (dichlorodiphenyltrichloroethane) is a colorless crystalline substance which is nearly insoluble in water but highly soluble in fats and most organic solvents. DDT is created by the reaction of trichloroethanol with [chlorobenzene] (C6H5CL). Trade or other names for DDT include Anofex, Cesarex, Chlorophenothane, Dedelo, Dichlorodiphenyltrichloroethane (DDT), Dinocide, Didimac, Digmar, ENT 1506, Genitox, Guesapon, Guesarol, Gexarex, Gyron, Hildit, Ixodex, Kopsol, Neocid, OMS 16, Micro DDT 75, Pentachlorin, Rukseam, R50 and Zerdane (Turosov et al., 2002). 1.1.1 ORIGIN AND HISTORY OF DDT: DDT was first synthesized in 1873, but its insecticidal properties were not discovered until 1939, by the Swiss scientist Paul Hermann Mller, who was awarded the 1948 No bel Prize in Physiology and Medicine for his efforts. DDT is the best-known of a number of chlorine-containing pesticides used in the 1940s and 1950s. It was used extensively during World War II by Allied troops and certain civilian populations to control insect typhus and malaria vectors (as a result nearly eliminating typhus). Civilian suppression used a spray on interior walls, which kills mosquitoes that rest on the wall after feeding to digest their meal. Resistant strains are repelled from the area. Entire cities in Italy were dusted to control the typhus carried by lice. DDT also sharply reduced the incidence of biting midges in Great Britain. DDT was responsible for eradicating malaria from Europe and

North America. Though today malaria is thought of as a tropical disease, it was more widespread prior to an extensive malaria eradication program carried out in the 1950s. Though this program was highly successful worldwide (reducing mortality rates from 192 per 100,000 to a low of 7 per 100,000), it was less effective in tropical regions due to the continuous life cycle of the parasite and poor infrastructure. It was not pursued aggressively in sub Saharan Africa due to perceived difficulties, with the result that mortality rates there were never reduced to the same dramatic extent, and now constitute the bulk of malarial deaths worldwide, especially following the resurgence of the disease as a result of microbe resistance to drug treatments and the spread of the deadly malarial variant caused by Plasmodium falciparum. DDT was also extensively used as an agricultural insecticide after 1945. DDT spraying in agricultural contexts was often orders of magnitudes greater in quantity than that employed for public health purposes, which required as little as 2g/m2 of DDT; by comparison, a single cotton field may have used a ton of DDT. By the 1950s, in some uses, doses of DDT and other insecticides had to be doubled or tripled as resistant insect strains developed. In addition, the evidence began to grow that the chemical became more concentrated at higher levels in the food chain (lundholm, 1997). 1.1.2 CHEMISTRY OF DDT: DDT is an organochlorine, similar in structure to the insecticide methoxychlor and the acaricide dicofol. It is a highly hydrophobic, colorless, crystalline solid with a weak, chemical odor. It is nearly insoluble in water but has a good solubility in most organic solvents, fats, and oils. DDT does not occur naturally, but is produced by the reaction of chloral (CCL13CHO) with chlorobenzene (C6H5CL) in the presence of sulfuric acid, which acts as a catalyst. Commercial DDT is a mixture of several closely related compounds. Dichlorodiphenyldichloroethylene (DDE) and Dichlorodiphenyldichloroethane (DDD) make up the balance. DDE and DDD

are also the major metabolites and breakdown products in the environment. The term TOTAL DDT is often used to refer to the sum of all DDT related compounds (DDT, DDE, and DDD) in a sample (lundholm, 1997). 1.2 APPLICATIONS OF DDT: DDT does not occur naturally in our

environment. It is applied by humans, for different purposes. As a pesticide, DDT was first used during World War II. It was as effective as an insect killer that some called it the "atomic bomb" of pesticides. After World War II, we realized that DDT could also be used on farms to control some common agricultural pests. Some of agricultural pests controlled by DDT: 1. Various potato beetles 2. Coddling moth (which attacks apples) 3. Corn earworm 4. Cotton bollworm 5. Tobacco budworms In addition to its use in farming, DDT was still used to control certain insects which carried diseases like malaria and yellow fever. Because of all these uses for DDT, the United States used a lot of it during the mid-1900s (lundholm, 1997). 1.2.1 APPLICATION TO THE ENVIRONMENT: Our environment is our surrounding. This includes living and non-living things around us. The nonliving components of environment are land, water and air. The living components are germs, plants, animals and people. All plants and animals adjust to the environment in which they are born and live. A change in any component of the environment may cause discomfort and affect normal life. Any unfavorable change or degeneration in the environment is known as Environmental Pollution. Environment is constituted by the interacting systems


of physical, biological and cultural elements inter-related in various ways, individually as well as collectively. DDT is present at many waste sites, including NPL sites (National Priorities List, and are the sites targeted for longterm federal cleanup activities). Releases from these sites might continue to contaminate the environment. Most DDT in the environment is a result of past use. DDT still enters the environment because of its current use in other areas of the world. DDE is only found in the environment as a result of contamination or breakdown of DDT. DDD also enters the environment during the breakdown of DDT. Large amounts of DDT were released into the air and on soil or water when it was sprayed on crops and forests to control insects. DDT was also sprayed in the environment to control mosquitoes. Although the use of DDT is no longer permitted in the United States, DDT may still be released into the atmosphere in other countries where it is still manufactured and used, including Mexico. DDT may also enter the air when they evaporate from contaminated water and soil. DDT in the air will then be deposited on land or surface water. This cycle of evaporation and deposition may be repeated many times. As a result, DDT can be carried long distances in the atmosphere. These chemicals have been found in bogs, snow, and animals in the Arctic and Antarctic regions, far from where they were ever used. Some DDT may have entered the soil from waste sites. DDT may occur in the atmosphere as a vapor or be attached to solids in air. Vapor phase DDT may break down in the atmosphere due to reactions caused by the sun. As a result, the half-life of these chemicals in the atmosphere as vapors (the time it takes for one-half of the chemical to turn into something else) is approximately 1.53 days. DDT last in the soil for a very long time. Eventually, most DDT breaks down into DDE and DDD, generally by the action of microorganisms. DDE and DDD also last in soil for long periods. These chemicals may also evaporate into the air and be deposited in other places. They stick strongly to soil, and therefore generally remain in the


surface layers of soil. Some soil particles with attached DDT may get into rivers and lakes in runoff. Only a very small amount, if any, will seep into the ground and get into groundwater. The length of time that DDT will last in soil depends on many factors including temperature, type of soil, and whether the soil is wet. DDT lasts for a much shorter time in the tropics where the chemical evaporates faster and where microorganisms degrade it faster. DDT disappears faster when the soil is flooded or wet than when it is dry. DDT disappears faster when it initially enters the soil. Later on, evaporation slows down and some DDT moves into spaces in the soil that are so small that microorganisms cannot reach the DDT to break it down efficiently. In tropical areas, DDT may disappear in much less than a year. In temperate areas, half of the DDT initially present usually disappears in about 5 years. However, in some cases, half of the DDT initially present will remain for 20, 30, or more years. In surface water, DDT will bind to particles in the water, settle, and be deposited in the sediment. DDT is taken up by small organisms and fish in the water. It accumulates to high levels in fish and marine mammals (such as seals and whales), reaching levels many thousands of times higher than in water. In these animals, the highest levels of DDT are found in the fat. DDT in soil can also be absorbed by some plants and by the animals or people who eat those crops (Benvenue, 1976). 1.2.2 APPLICATION AS AN INSECTICIDE: An insecticide is a pesticide used against insects. They include ovicides and larvicides used against the eggs and larvae of insects respectively. Insecticides are used in agriculture, medicine, industry and the household. The use of insecticides is believed to be one of the major factors behind the increase in agricultural productivity in the 20th century. Nearly all insecticides have the potential to significantly alter ecosystems. Many are toxic to humans, and others are concentrated in the food chain. The classification of insecticides is done in several different ways

1. Systemic insecticides are incorporated by treated plants. Insects ingest

the insecticide while feeding on the plants.


Contact insecticides are toxic to insects brought into direct contact. Efficacy is often related to the quality of pesticide application, with small droplets (such as aerosols) often improving performance.


Natural insecticides, such as nicotine, pyrethrum and neem extracts are made by plants as defenses against insects. Nicotine based insecticides are still being widely used in the US and Canada though they are barred in the EU.

4. Plant-incorporated






produced by plants after genetic modification. For instance, a gene that codes for a specific Baccilus thuringiensis biocidal protein is introduced into a crop plant's genetic material. Then, the plant manufactures the protein. Since the biocide is incorporated into the plant, additional applications at least of the same compounds are not required.
5. Inorganic insecticides are manufactured with metals and include

arsenates, copper compounds and fluorine compounds, which are now seldom used, and sulfur, which is commonly used.
6. Organic insecticides are synthetic chemicals which comprise the largest

numbers of pesticides available for use today.

7. Mode of action: How the pesticide kills or inactivates a pest is another

way of classifying insecticides. Mode of action is important in predicting whether an insecticide will be toxic to unrelated species, such as fish, birds and mammals.

DDT is an organochloride. It was introduced as a safer alternative to the lead and arsenic compounds. Some insecticides have been banned due to the fact that

they are persistent toxins which have adverse effects on animals and, or humans. An often quoted case is that of DDT, an example of a widely used pesticide, which was brought to public attention by Rachel Carson's book, Silent Spring. One of the better known impacts of DDT is to reduce the thickness of the egg shells on predatory birds. The shells sometimes become too thin to be viable, causing reductions in bird populations. This occurs with DDT and a number of related compounds due to the process of bioaccumulation, wherein the chemical, due to its stability and fat solubility, accumulates in organisms fatty tissues. Also, DDT may biomagnify, which causes progressively higher concentrations in the body fat of animals farther up the food chain. The nearworldwide ban on agricultural use of DDT and related chemicals has allowed some of these birds, such as the peregrine falcon, to recover in recent years. A number of the organochlorine pesticides have been banned from most uses worldwide, and globally they are controlled through the Stockholm Convention on persistent organic pollutants. These include aldrin, chlordane, DDT, dieldrin, endrin, heptachlor, mirex and toxaphene (van, et al., 1996). 1.2.3 APPLICATION FOR DISEASE CONTROL: Malaria infects between 300 million and 500 million people every year. The World Health Organisation estimates that around 1 million people die from malaria every year. Most of those deaths (90%) occur in Africa and mostly in children under the age of 5. The economic impact includes costs of health care, working days lost to sickness, days lost in education, decreased productivity due to brain damage from cerebral malaria, and loss of investment and tourism (Tren et al., 2004). Most of the prior use of DDT was in agriculture. Current use for disease control requires only a small fraction of the amounts used previously and is much less likely to cause environmental problems. Such limited use of DDT has not become ineffective due to resistance in areas where it is used inside homes. There are insecticide alternatives to DDT, and Vietnam is often mentioned as a

country that has seen a continued decline in malaria cases after involuntarily switching from DDT in 1991. However, Vietnam's neighbor Thailand has continued to use DDT and has a much smaller malaria rate despite similar conditions. The insecticide alternatives are generally more expensive, which limits their use in poor nations and in situations where anti-malarial efforts are already underfunded. It is doubtful that they are more environmentally friendly or as efficient, easy to use and safe for humans as DDT. In many African nations, the problems resulting from malaria are viewed as greater than the potential dangers of DDT. After South Africa stopped using DDT in 1996, the number of malaria cases in KwaZulu Natal province rose from 8,000 to 42,000 cases. By 2000, there had been an approximate 100 percent increase in malaria deaths. Today, thanks to DDT, the number of deaths from malaria in the region is less than 50. South Africa could afford and did try newer alternatives to DDT but they proved less effective (Tren et al., 2004). 1.3 EFFECT OF DDT: DDT is an organochloride insecticide. It is a persistent environmental contaminant and its widespread use has resulted in worldwide contamination. 1.3.1 EFFECT DUE TO DDT TRANSPORT: People are exposed to DDT mainly by eating foods containing small amounts of these compounds. Even though DDT has not been used in this country since 1972, soil may still contain some DDT that may be taken up by plants and eaten by animals and people. DDT from contaminated water and sediment may be taken up by fish. The amount of DDT in food has greatly decreased since DDT was banned and should continue to decline. In the years 1986 to 1991, the average adult in the United States consumed an average of 0.8 micrograms (a microgram is a millionth of a gram) of DDT a day. Adults consumed slightly different amounts based on their age and sex. The largest fraction of DDT in a persons diet comes from meat, fish, poultry, and dairy products. Leafy vegetables generally contain

more DDT than other vegetables, possibly because DDT in the air is deposited on the leaves. Infants may be exposed by drinking breast milk. DDT or its breakdown products are still present in some air, water, and soil samples. However, levels in most air and water samples are presently so low that exposure is of little concern. DDT levels in air have declined to such low levels that it often cannot be detected. In cases where DDT has been detected in air, it is associated with air masses coming from regions where DDT is still used or from the evaporated DDT from contaminated water or soil. DDT concentrations measured in air in the Great Lakes region in 1990 reached maximum levels of 0.035 and 0.119 nanograms (a nanogram is a billionth of a gram) of chemical per cubic meter of air (ng/m3), respectively. Levels were generally much lower, especially during the winter months. In 19951996, soils in the Corn Belt, where DDT was heavily used in the past, contained on the average about 10 nanograms of DDT in a gram of soil. In recent years, most surface water has not contained detectable amounts of DDT. People who work or live around NPL sites (National priorities list) or work with contaminated soil or sediment would most likely be exposed by accidentally swallowing soil, having skin contact with the soil, or breathing in DDT in dust (Bevenue, 1976) 1.3.2 EFFECT DUE TO DDT METABOLISM: DDT enters the body mainly when a person eats contaminated food. The actual amount of DDT absorbed from foods depends on both the concentration of chemical in the food and the amount of food eaten. Small amounts of DDT may also be breathed in and absorbed into the body. DDT is often attached to particles too large to pass very far into the lungs after air containing them is breathed. These particles are more likely to be carried upward in the mucus of the air passages and swallowed than for the DDT to be absorbed in the lungs. DDT does not enter the body through the skin very easily. Once inside the body, DDT can break down to DDE or DDD. DDE and DDD, in turn, break down to other substances (called metabolites). DDT, DDE, and DDD are stored most readily in fatty tissue.

Stored amounts leave the body very slowly. Levels in fatty tissues may either remain relatively the same over time or even increase with continued exposure. However, as exposure decreases, the amount of DDT in the body also decreases. DDT metabolites leave the body mostly in urine, but may also leave by breast milk (Adelercreuty, 1995). 1.3.3 DDT EFFECT ON HEALTH: Eating food with large amounts of DDT over a short time would most likely affect the nervous system. People who swallowed large amounts of DDT became excitable and had tremors and seizures. They also experienced sweating, headache, nausea, vomiting, and dizziness. These effects on the nervous system went away once exposure stopped. Tests in laboratory animals confirm the effect of DDT on the nervous system. No effects have been reported in people given small daily doses of DDT by capsule for 18 months. People exposed for a long time to small amounts, such as people who worked in factories where DDT was made, had some reversible changes in the levels of liver enzymes in the blood. To protect the public from the harmful effects of toxic chemicals and to find ways to treat people who have been harmed, scientists use many tests. One way to see if a chemical will hurt people is to learn how the chemical is absorbed, used, and released by the body, for some chemicals, animal testing may be necessary. Animal testing may also be used to identify health effects such as cancer or birth defects. Without laboratory animals, scientists would lose a basic method to get information needed to make wise decisions to protect public health. Scientists have the responsibility to treat research animals with care and compassion. Laws today protect the welfare of research animals, and scientists must comply with strict animal care guidelines. Animal studies show that longterm exposure to DDT may affect the liver. Tests in animals also suggest that short-term exposure to DDT and metabolites in food may have a harmful effect on reproduction. In addition, we know that some breakdown products of DDT can cause harmful effects on the adrenal gland. This gland is situated near the

kidney and produces hormones. Studies in animals have shown that oral exposure to DDT can cause liver cancer. Studies of DDT-exposed workers did not show increases in deaths or cancers. The Department of Health and Human Services has determined that DDT may reasonably be anticipated to be a human carcinogen. The International Agency for Research on Cancer (IARC) has determined that DDT may possibly cause cancer in humans. Environmental Protection Agency has determined that DDT is probable human carcinogens (Bouwman et al., 1992). 1.3.4 DDT EFFECT ON CHILDREN: This discusses potential health effects from exposures during the period from conception to maturity at 18 years of age in humans. Potential effects on children resulting from exposures of the parents are also considered. Children can be exposed to DDT by eating food contaminated with these compounds. DDT is a pesticide, and even though it has not been used in this country since 1972, soil has small amounts and, under certain conditions, contaminated soil transfers DDT to crops (Albone et al., 1972). Children can be exposed also by eating food imported from countries where DDT is still being used. Because of their smaller weight, childrens intake of DDT per kilogram of body weight may be greater than that of adults. In the United States between 1985 and 1991, the average 8-month-old infant consumed 4 times as much DDT for each pound of body weight than the average adult. However, the amounts of DDT consumed were very small. DDT from the mother can enter her unborn baby through the placenta. DDT has been found in human placentas, fetuses, and umbilical cord blood. Because DDT has been measured in human milk, nursing infants are also exposed to DDT. However, in most cases, the benefits of breastfeeding outweigh any risks from exposure to DDT in mothers milk. We do not know whether children differ from adults in their susceptibility to health effects from DDT. There have been few studies of health effects in young children exposed to DDT. A child who

drank DDT in kerosene vomited and had tremors and convulsions and eventually died; however, we do not know how much of this was caused by the kerosene. Adults who swallowed DDT in much greater amounts than those found in the environment had effects on their nervous systems. The same harmful effects will probably happen to young children if they eat food or drink liquids with large amounts of DDT. However, because DDT is no longer used or made in the United States, such exposure is not likely to happen. Two studies have shown a higher dose of DDT is needed to kill newborn and young rats than adult rats. In one study, when the dose was divided up and given over four days, the same dose of DDT killed rats of all ages. There is no evidence that exposure to DDT at levels found in the environment causes birth defects or other developmental effects in people. (Adeshina, 1991), Studies in animals have shown that DDT given during pregnancy can slow the growth of the fetus, but there is no evidence that exposure to DDT causes structural birth defects in animals. However, exposure to DDT or its metabolites during development may change how the reproductive and nervous systems work. Male rats exposed to the DDT breakdown product, DDE, as fetuses or while nursing, showed changes in the development of their reproductive system. One study found that the beginning of puberty is delayed in male rats given relatively high amounts of DDE as juveniles. Also, one study showed that exposure of mice to DDT during the first weeks of life resulted in neurobehavioral problems when tests were done later in life. These studies raise concerns that exposure to DDT early in life might cause harmful effects that remain or begin long after exposure has stopped. (Agarwal et al., 1978)








Experimental work suggests that some species, notably bats, may have been affected by DDT and its metabolites. Species which show marked seasonal

cycles in fat content are most vulnerable, but few experimental studies on such species have been made. In contrast to the situation in birds, where the main effect of DDT is on reproduction, the main known effect in mammals is to increase the mortality of migrating adults. The lowest acute dose which kills American big brown bats is 20 mg/kg. Bats collected from the wild (and containing residues of DDE in fat) die after experimental starvation, which simulates loss of fat during migration (Tuckey, 1971). 1.3.6 DDT TOXICITY TO MICROORGANISM: Aquatic microorganisms are more sensitive than terrestrial ones to DDT. An environmental exposure concentration of 0.1 g/litre can cause inhibition of growth and photosynthesis in green algae. Repeated applications of DDT can lead to the development of tolerance in some microorganisms. There is no information concerning the effects on species composition of microorganism communities. Therefore, it is difficult to extrapolate the relevance of single-culture studies to aquatic or terrestrial ecosystems. However, since microorganisms are basic in food chains, adverse effects on their populations would influence ecosystems. Thus, DDT and its metabolites should be regarded as a major environmental hazard (Tuckey, 1971). 1.3.7 DDT TOXICITY TO AQUATIC INVERTIBRATEST: Both the acute and long-term toxicities of DDT vary between species of aquatic invertebrates. Early developmental stages are more sensitive than adults to DDT. Long-term effects occur after exposures to concentrations ten to a hundred times lower than those causing short-term effects. DDT is highly toxic, in acute exposure, to aquatic invertebrates at concentration as low as 0.3g/litre. Toxic effects include impairment of reproduction and development, cardiovascular modifications, and neurological changes. Daphnia reproduction is adversely affected by DDT at 0.5g/litre. The influence of environmental variables (such as temperature, water hardness, etc.) is documented but the mechanism

is not fully understood.

In contrast to the data on DDT, there is little

information on the metabolites DDE. The reversibility of some effects, once exposure ceases, and the development of resistance have been reported (Tuckey, 1971). 1.3.8 DDT EFFECT ON EGG SHELL: The alleged thinning of eggshells by DDT in the diet was effective propaganda. However, actual feeding experiments proved that there was very little, if any, correlation between DDT levels and shell thickness. Thin shells may result when birds are exposed to fear, restraint, mercury, lead, parathion, or other agents, or when deprived of adequate calcium, phosphorus, Vitamin D, light, calories, or water. While quail fed a diet containing 2 percent calcium produced thick shells, a calcium content of only 1 percent resulted in shells 9 percent thinner than normal (Tucker, 1971). In the presence of lead, shells were 14 percent thinner, and with mercury, 8 percent thinner (Tuckey, 1971). Bitman and co workers demonstrated eggshell thinning with DDT by reducing calcium levels to 0.56 percent from the normal 2.5 percent. After this work was exposed as anti-DDT propaganda, Bitman continued his work for another year. Instead of the calcium-deficient diets, however, he fed the quail 2.7 percent calcium in their food. The shells they produced were not thinned at all by the DDT. Unfortunately, the editor of science refused to publish the results of that later research. Editor Philip Abelson had already told Dr. Thomas Jukes of the University of California in Berkeley that science would never publish anything that was not antagonistic toward DDT (T. Jukes, personal communication). Bitman therefore had to publish the results of his legitimate feeding experiments in an obscure specialty journal (Bitman, 1971), and many readers of science continued to believe that DDT could cause birds to lay thin egg shell. 1.4 FATE OF DDT TO THE ENVIRONMENT: DDT and its metabolites may be transported from one medium to another by the processes of

solubilization, adsorption, remobilization, bioaccumulation, and volatilization. In addition, DDT can be transported within a medium by currents, wind, and diffusion. Organic carbon partition coefficients of 1.5x105 (Swann et al. 1981), reported for DDT suggest that this compound adsorb strongly to soil. This chemical is only slightly soluble in water, with solubility of 0.025 mg/L (Howard et al., 1997). Therefore, loss of this compound in runoff is primarily due to transport of particulate matter to which these compound is bound. There is evidence that DDT, as well as other molecules, undergoes an aging process in soil whereby the DDT is sequestered in the soil so as to decrease its bioavailability to microorganisms, extractability with solvents, and toxicity to some organisms (Alexander 1995), (Peterson et al. 1971), (Robertson et al.,1998). At the same time, analytical methods using vigorous extractions do not show significant decreases in the DDT concentration in soil. In one such study, DDT was added to sterile soil at various concentrations and allowed to age (Robertson et al., 1998). At intervals, the toxicity of the soil was tested using the house fly, fruit fly, and German cockroach. After 180 days, 84.7% of the insecticide remained in the soil, although more than half of the toxicity had disappeared when the fruit fly was the test species, and 90% had disappeared when the house fly was the test species. The effect with the German cockroach was not as marked. Recently, a study was conducted to determine the bioavailability of DDT, DDE, and DDD to earthworms (Morrison et al. 1999). It was shown that the concentrations of DDT, DDE, DDD, and DDT were consistently lower in earthworms exposed to these compounds that had persisted in soil for 49 years than in earthworms exposed to soil containing freshly added insecticides at the same concentration. The uptake percentages of DDT and its metabolites by earthworms were in the range of 1.301.75% for the 49-year-aged soil, but were 4.0015.2% for the fresh soil (Morrison et al. 1999). Long term monitoring data have also indicated that aged and sequestered DDT are not subject to significant volatilization, leaching, or degradation (Boul

et al. 1994). The concentrations of DDT, DDE, and DDD monitored at two sites in a silt loam in New Zealand declined from 1960 to 1980, but very little loss was evident from 1980 to 1989 (Boul et al. 1994). The lack of appreciable biodegradation as DDT ages in soil suggests that the compound is not bioavailable to microorganisms. Aging is thought to be associated with the continuous diffusion of a chemical into micropores within soil particles where it is sequestered or trapped, and is therefore unavailable to microorganisms, plants, and animals (Alexander 1995). In the case of biodegradation, the aging process results in the gradual unavailability of substrate that makes the reaction kinetics appear to be nonlinear. There is abundant evidence that DDT gets into the atmosphere as a result of emissions or volatilization. The process of volatilization from soil and water may be repeated many times and, consequently, DDT may be transported long distances in the atmosphere by what has been referred to as a global distillation from warm source areas to cold Polar Regions. As a result, DDT and its metabolites are found in arctic air, sediment, and snow with substantial accumulations in animals, marine mammals, and humans residing in these regions (Harner, 1997). An analysis of sediment cores from eight remote lakes in Canada indicated that DDT concentrations in surface sediments (01.3 cm depth) declined significantly with latitude (Muir et al., 1995). The maximum DDT concentrations in core slices in midcontinent lakes date from the late 1970s to 1980s, which is about 510 years later than the maximum for Lake Ontario. Volatilization of DDT, DDE, and DDD is known to account for considerable losses of these compounds from soil surfaces and water. Their tendency to volatilize from water can be predicted by their respective Henry's law constants, which for the respective p,p- and o,p- isomers are 8.3x10-6, 2.1x10-5, 4.0x106, 5.9x10-7, 1.8x10-5, and 8.2x10-6 atm-m3/mol (Howard et al.,1997). The predicted volatilization half-lives from a model river 1m deep, flowing at 1m/sec, with a wind of 3m/sec are 8.2, 3.3, 10.5, 6.3, 3.7, and 8.2 days,

respectively. Laboratory studies of the air/water partition coefficient of DDE indicate that it will volatilize from seawater 1020 times faster than from freshwater (Atlas et al., 1982). Volatilization from moist soil surfaces can be estimated from the Henrys law constant divided by the adsorptivity to soil (Dow Method) (Thomas 1990). The predicted half-life for DDT volatilizing from soil is 23 days, compared to an experimental half-life of 42 days. (Sleicher et al.,1984),estimated a volatilization half-life of 110 days for DDT from soil in Kenya based on mass transfer through the boundary layers, and claimed that volatilization of DDT was sufficient to account for its rapid disappearance from soil. However, laboratory experiments in which p,p-DDT was incubated in an acidic (pH 4.54.8), sandy loam soil maintained at 45 EC for 6 hours/day for 6 weeks resulted in neither volatilization of DDT or its metabolites nor mineralization (Andrea et al., 1994). Other studies using a latosol soil (pH 5.7) found that 5.9% of the radioactivity was lost through volatilization during 6 week incubation at 45 EC (Sjoeib et al., 1994). The volatilization rate of DDT from soil is significantly enhanced by temperature, sunlight, and flooding of the soil (Samuel et al., 1990). DDT is removed from the atmosphere by wet and dry deposition and diffusion into bodies of water. The largest amount of DDT is believed to be removed from the atmosphere in precipitation (Woodwell et al., 1971). DDT, DDE, and DDD are highly lipid soluble, as reflected by their log octanolwater partition coefficients of 6.91, 6.51, and 6.02, respectively for the p,pisomers and 6.79, 6.00, and 5.87, respectively for the o,p- isomers (Howard and Meylan, 1997). This lipophilic property, combined with an extremely long half-life is responsible for its high bioconcentration in aquatic organisms (levels in organisms exceed those levels occurring in the surrounding water). Organisms also feed on other animals at lower trophic levels. The result is a progressive biomagnification of DDT in organisms at the top of the food chain. (Biomagnification is the cumulative increase in the concentration of a persistent

contaminant in successively higher trophic levels of the food chain (from algae to zooplankton to fish to birds). (Ford et al., 1991) reported increased biomagnification of DDT, DDE, and DDD from soil sediment to mosquito fish, a secondary consumer. No distinct pattern of biomagnification was evident in other secondary consumers such as carp and small mouth buffalo fish. The biomagnification of DDT is exemplified by the increase in DDT concentration in organisms representing four trophic levels sampled from a Long Island estuary. The concentrations in plankton, invertebrates, fish, and fish-eating birds were 0.04, 0.3, 4.1, and 24 mg/kg, whole body basis (Leblanc, 1995). (Evans et al., 1991) reported that DDE biomagnified 28.7 times in average concentrations from plankton to fish and 21 times from sediment to amphipods in Lake Michigan. In some cases, humans may be the ultimate consumer of these contaminated organisms. The bioconcentration factor (BCF) is defined as the ratio of the equilibrium concentration of contaminant in tissue compared to the concentration in ambient water, soil, or sediment to which the organism is exposed. There are numerous measurements and estimates of BCF values for DDT in fish. Oliver and Niimi (1985) estimated the steady-state BCF in rainbow trout as 12,000. Other BCF values that have been reported include 51,000100,000 in fish, 4,550690,000 in mussels, and 36,000 in snails (Davies et al.,1984), (Geyer et al., 1982), (Metcalf, 1973) (Reish et al., 1978), Veith et al., 1979). DDT bioconcentration studies in aquatic environments with representatives of various trophic levels demonstrate that bioconcentration increases with increasing trophic level (LeBlanc, 1995). Trophic level differences in bioconcentration are largely due to increased lipid content and decreased elimination efficiency among higher level organisms. However, biomagnification also contributes to the increased concentration of DDT in higher trophic organisms (LeBlanc, 1995). Fish move from the Great Lakes or other bodies of water with elevated DDT levels to rivers that feed into these lakes. In doing so, they transport DDT, which may represent a risk to wildlife

along the tributaries (Giesy et al,. 1994). Despite being strongly bound to soil, at least a portion of DDT, DDE, and DDD is bioavailable to plants and soil invertebrates. (Nash et al., 1970) studied the DDT residues in soybean plants resulting from the application of DDT to the surface or subsurface soil. They found that the major source of DDT contamination was due to sorption of volatilized residues from surface-treated soil. This was 6.8 times greater than that obtained through root uptake and translocation after subsurface treatment. In other experiments with oats and peas, root uptake of DDT was low and there was little or no evidence of translocation of the insecticide (Fuhremann et al., 1980), and (Lichtenstein et al., 1980). (Verma et al., 1991) reported that grain, maize, and rice plants accumulate DDT adsorbed to soil. Most of the residues were found in the roots of the plant, and the lowest concentration of DDT residues was found in the shoots, indicating low translocation of DDT. Earthworms are capable of aiding the mobilization of soil-bound DDT residues to readily bioavailable forms (Verma et al., 1991). DDT may collect on the leafy part of plants from the deposition of DDT-containing dust. 1.5 BIODEGRADATION OF DDT: Biodegradation or biotic degradation or biotic decomposition is the chemical dissolution of materials by bacteria or other biological means. The term is often used in relation to ecology, waste management, biomedicine, and the natural environment (Bioremediation) and is now commonly associated with environmentally friendly products that are capable of decomposing back into natural elements. Organic material can be degraded aerobically with oxygen, or anaerobically, without oxygen. A term related to biodegradation is biomineralisation, in which organic matter is converted into minerals. Biosurfactant, an extracellular surfactant secreted by microorganisms, enhances the biodegradation process. Biodegradable matter is generally organic material such as plant and animal matter and other substances originating from living organisms, or artificial materials that are similar enough to plant and animal matter to be put to use by microorganisms. Some

microorganisms have a naturally occurring, microbial catabolic diversity to degrade, transform or accumulate a huge range of compounds including hydrocarbons (Example oil), polychlorinated biphenyls (PCBs), polyaromatic hydrocarbons (PAHs), pharmaceutical substances, radionuclides and metals. Major methodological breakthroughs in microbial biodegradation have enabled detailed genomic, metagenomic, proteomic, bioinformatic and other highthroughput analyses of environmentally relevant microorganisms providing unprecedented insights into key biodegradative pathways and the ability of microorganisms to adapt to changing environmental conditions. Products that contain biodegradable matter and non-biodegradable matter are often marketed as biodegradable (Aislabie et al., 1997). 1.5.1: BIODEGREDATION OF DDT IN AIR: In the atmosphere, about 50% of DDT is adsorbed to particulate matter and 50% exists in the vapor phase (Bidleman, 1988). In the vapor phase, DDT reacts with photochemically produced hydroxyl radicals with an estimated rate constant of 3.44x10-12 cm3/molecule-sec determined from a fragment constant estimation method (Meylan and Howard 1993). Assuming an average hydroxyl radical concentration of 1.5x106 per cm3, its half-life will be 37 hours. Both DDE and DDD have higher vapor pressures than DDT, and a smaller fraction of these compounds will be adsorbed to particulate matter. The estimated half-lives of vapor-phase DDE and DDD are 17 and 30 hours, respectively. Direct photolysis may also occur in the atmosphere. DDT, DDE, and DDD adsorbed on particulate matter are not expected to undergo photooxidation rapidly, and therefore, may be subject to long-range transport. When atmospheric sampling of pesticides was performed at nine localities in the United States during a time of high DDT usage, DDT was mostly present in the particulate phase (Stanley et al. 1971). 1.5.2: BIODEGREDATION OF DDT IN WATER: DDT, DDE, and DDD present in water may be transformed by both photodegradation and

biodegradation. Since the shorter wave radiation does not penetrate far into a body of water, photolysis primarily occurs in surface water and is dependent on the clarity of the water. Direct photolysis of DDT and DDD are very slow in aquatic systems, with estimated half-lives of more than 150 years. Direct photolysis of DDE results in a half-life of about 1 day in summer and 6 days in winter. DDE also undergoes photoisomerization when exposed to sunlight. Photolysis of DDE photoisomers is slower by at least one order of magnitude compared to DDE. Indirect photolysis of DDT appears to be rapid in some natural waters. In one study, 50% of DDT was lost in San Francisco Bay water after 7 days of exposure to sunlight. No DDE or DDD photoproducts were found, although DDE would be expected to be produced based on photolysis studies with the DDT analog, methoxychlor, in several freshwaters. This may reflect different mechanisms in natural waters containing different photosensitizers. Studies with DDT at shorter wavelengths suggest that the initial reaction results in the dissociation of the C12CCl bond. No information on the indirect photolysis of DDE or DDD was located (Coulston, 1985), (Zepp et al., 1977). Photoinduced addition of DDT to a model lipid, methyl oleate, indicates that light-induced additions of DDT to unsaturated fatty acids of plant waxes and cutins may occur on a large scale (Schwack, 1988). DDT undergoes hydrolysis by a base-catalyzed reaction resulting in a half-life of 81 days at pH 9. Biodegradation of DDT in water is reported to be a minor mechanism of transformation (Johnsen, 1976). 1.5.3: BIODEGREDATION OF DDT IN SEDIMENTS AND SOIL: Four mechanisms have been suggested to account for most losses of DDT residues from soils. They are volatilization, removal by harvest (Example plants that have absorbed the residue), water runoff, and chemical transformation (Fishbein, 1973). Three of these are transport processes, and the fourth, chemical transformation, may occur by abiotic and biotic processes. Photooxidation of DDT is known to occur on soil surfaces or when adsorbed to

sediment (Baker et al., 1970), (Lichtenstein et al.,1959), (Miller et al.,1979). The conversion of DDT to DDE in soil was enhanced by exposure to sunlight in a 90-day experiment with 91% of the initial concentration of DDT remaining in the soil for an unexposed dark control and 65% remaining for the sample exposed to light (Racke et al., 1997). However, UV-irradiation of DDT on soil for 10 hours mineralized less than 0.1% of the initial amount (Mineralization is the complete degradation of a chemical, generally to carbon dioxide and water for an organic chemical containing carbon, hydrogen, and oxygen) (Vollner et al., 1994). The amount of DDT that may have been converted to DDE was not reported. Biodegradation may occur under both aerobic and anaerobic conditions due to soil microorganisms including bacteria, fungi, and algae (Arisoy, 1998), (Lichtenstein et al., 1959), (Menzie, 1980), (Stewart et al., 1971), and (Verma et al., 1991). Since biodegradation studies generally focus on the loss of the parent compound rather than complete degradation or mineralization, and since DDT initially biodegrades to DDD or DDE, there still may be dangerous compounds after almost all of the DDT that was originally present has biodegraded. During biodegradation of DDT both DDE and DDD are formed in soils. Both metabolites may undergo further transformation but the extent and rate are dependent on soil conditions and, possibly, microbial populations present in soil. The degradation pathways of DDT under aerobic and anaerobic conditions have been reviewed by Zook et al., 1999) and (Aislabie et al., 1997). Ligninolytic or lignindegrading fungi have been shown to possess the biodegradative capabilities for metabolizing a large variety of persistent compounds, including DDT. Mineralization of DDT was even observed in laboratory experiments using a member of this group of fungi, Phanerochaete chrysosporum (a white rot fungus) (Aislabie et al., 1997). Biodegradation of DDT and its metabolites involves cometabolism, a process in which the microbes derive nutrients for growth and energy from sources other than the

compound of concern. DDE, the dominant DDT metabolite found, is often resistant to biodegradation under aerobic and anaerobic conditions (Strompl et al., 1997). Recent laboratory experiments in marine sediment showed that DDE is dechlorinated to DDMU (1-chloro-2,2-bis(p-chlorophenyl)ethylene) under methanogenic or sulfidogenic conditions (Quensen et al., 1998). DDD is also converted to DDMU, but at a much slower rate. DDMU degrades further under anaerobic conditions. No evidence was found that methylsulfonyl metabolites of DDT are formed as a result of microbial metabolism. The rate at which DDT is converted to DDD in flooded soils is dependent on the organic content of the soil (Racke et al. 1997). In a laboratory study, Hitch and Day (1992) found that soils with a low metal content degrade DDT to DDE much more slowly than do soils with high metal content. As mentioned earlier, the half-life represents the estimated time for the initial disappearance of 50% of the compound in question and does not necessarily imply that first-order kinetics were observed throughout the experiment unless otherwise noted. In the case of DDT, the disappearance rate slows considerably so that after the initial concentration is reduced by half, the time required for the loss of half of that which remains is substantially longer. This is largely because much of the initial loss of compound is due to volatilization, rather than biodegradation. Biodegradation rate slows in time because DDT migrates into micro pores in soil particles where it becomes sequestered and unavailable to soil microorganisms (Alexander, 1995, 1997). In addition, the disappearance of DDT is often reported as the disappearance of DDT residues, and therefore, the reported rate of loss is a summation of the component DDT-related chemicals. DDT breaks down into DDE and DDD in soil, and the parent-to-metabolite ratio (DDT to DDE or DDD) decreases in time. However, this ratio may vary considerably with soil type. In a 19951996 study of agricultural soils in the corn belt of the central United States, the ratio of DDT varied from 0.5 to 6.6 with threequarters of the soils having ratios above 1 (Aigner et al., 1998). In a study of

forest soils in Maine, the half-life for the disappearance of DDT residues was noted to be 2030 years. DDT was much more persistent in muck soils than in dry forest soils. A study of DDT in agricultural soils in British Colombia, Canada reported that over a 19-year period, there was a 70% reduction of DDT in muck soils and a virtual disappearance of DDT from loamy sand soils (Aigner et al., 1998). Land management practices also affect the persistence of DDT. In 1971, an experiment was conducted in a field containing high amounts of DDT to evaluate the effect of various management tools in the disappearance of the insecticide (Spencer et al., 1996). The site was revisited in 1994 to determine the residual concentrations of DDT and its metabolites and to measure volatilization fluxes. Concentrations of DDT were reduced in all plots and the major residue was DDE. The highest concentrations of residues were found in deep plowed and unflooded plots. Deep plowing places the DDT deeper into the soil profile, possibly reducing volatilization. The volatilization rate of DDT is enhanced by flooding the soil (Samuel et al., 1990). Under flooded, reducing conditions, DDD was a more common degradation product of DDT than DDE. Significant concentration of DDT was detected in the atmosphere over the plots. Irrigating the soil dramatically increased the volatilization flux of all DDT analogs. This is probably related to the amount of DDT in the soil solution. Volatilization, air transport, and redeposition were found to be the main avenues of contaminating forage eaten by cows. In microcosm experiments, (Boul, 1996) found that increasing soil water content enhanced DDT loss from generally aerobic soil. His results suggested that increased biodegradation contributed to these effects. (Boule et al., 1994) analyzed DDT residues in pasture soil as they were affected by long-term irrigation and super phosphate fertilizer application. They found that DDT residues in irrigated soil were about 40% that of unirrigated soil. The predominant residue was DDE, and these residues were much higher in unirrigated than in irrigated soil. DDE is lost at a lower rate than DDT. DDD

residues were very low in both irrigated and non irrigated soil indicating that loss of DDD must occur at a rate at least as great as it is generated from DDT. Super phosphate treatment, which is known to increase microbial biomass, also resulted in lower levels of DDT and DDT than in unfertilized controls. The distribution of DDT with depth suggests that irrigation did not cause increased leaching of the insecticide. A set of experiments was conducted during 19821987 and 19891993 in 14 countries under the auspices of the International Atomic Energy Agency (IAEA) on the dissipation of DDT from soil under field conditions in tropical and subtropical areas (Racke et al., 1997). After 12 months, the quantity of DDT and metabolites remaining in soil at tropical sites ranged from 5% of applied in Tanzania to 15% in Indonesia. The half-life of DDT ranged from 22 days in Sudan to 365 days in China. One exception was in an extremely acidic soil (pH 4.5) in Brazil in which the half-life was >672 days. The conclusion of the study was that DDT dissipated much more rapidly under tropical conditions than under temperate condition. The major mechanisms of dissipation under tropical conditions were volatilization, biological and chemical degradation, and to a lesser extent, adsorption. Comparable half-live in temperate region that have been reported ranges from 837 to 6,087 days (Lichtenstein et al., 1959), (Racke et al., 1997), and (Chisholm et al., 1971). One investigator concluded that the mean lifetime of DDT in temperate U.S. soils was about 5.3 years (Racke et al., 1997). The primary metabolite detected in tropical soil was DDE. With the exception of highly acidic soil from Brazil, the half-lives for DDE ranged from 151 to 271 days, much less than the 20 years reported for DDE in temperate areas. The increased dissipation of DDT in the tropics compared with that in temperate zones is believed to be largely due to increased volatility under tropical conditions (Racke et al., 1997). 1.6 CURRENT STATUS OF DDT: Since 1996, EPA has been participating in international negotiations to control the use of DDT and other persistent organic

pollutants used around the world. Under the auspices of the United Nations Environment Programme, countries joined together and negotiated a treaty to enact global bans or restrictions on persistent organic pollutants (POPs), which includes DDT, known as the Stockholm Convention on POPs. The Convention includes a limited exemption for the use of DDT to control mosquitoes which are vectors that carry malaria-a disease that still kills millions of people worldwide. In September 2006, the World Health Organization (WHO) declared its support for the indoor use of DDT in African countries where malaria remains a major health problem, citing that benefits of the pesticide outweigh the health and environmental risks. This is consistent with the Stockholm Convention on POPs, which bans DDT for all uses except for malaria control. DDT is one of 12 pesticides recommended by the WHO for indoor residual spray programs. It is up to countries to decide whether or not to use DDT. EPA works with other agencies and countries to advise them on how DDT programs are developed and monitored, with the goal that DDT be used only within the context of Integrated Vector Management programs, and that it be kept out of agricultural sectors. 1.7 AIMS AND OBJECTIVE: The aims and objective of this research are 1. To develop a defined microbial consortium that will be used for the degradation of DDT. 2. To determine the maximum degradation time of DDT. 3. To develop or optimize conditions for the degradation of DDT.

1.8 RESEARCH PROBLEM DDT has been very effective in killing or repelling mosquitoes, its use has been severely reduced and restricted to indoor residual spraying, due to its


persistence in the environment and ability to bio-concentrate in the food chain. This project is to give recommendation on the wise use of DDT if it can not be fully eradicated from use in the farms and homes. 1.9 SCOPE OF THE STUDY The scope of the study is limited to uncultivated farm lands in campus 3 site, Delta State University, Abraka. 1.10 LIMITATION OF STUDY During the course of this research work, the following constrain were experienced. Financial problem Problem of resource materials Lack of experts to advise and give a guide during the laboratory analysis Time constrain, the analysis took months before it was completed which was very challenging during the daily monitoring of the analysis. Problem with transportation since the laboratory was far from school.



2.0 COLLECTION OF SOIL SAMPLES: The first step was to determine the number of samples needed from the field. And this depends on the amount of variability within the field. Factors that were considered include: Soil types Soil texture Slopes Drainage Erosion The soil samples were collected from Delta State University, Campus 3 site, Abraka, Delta state. Before sampling, the dept or sampling dept was checked. Sampling the soil in an organized pattern is a good management practice, because it helps to ensure adequate representation of the entire field, and that was properly observed. The materials used for the soil sampling were:

Trowel (sterilized). Sample containers (plastic), which were sterile.

Stratified Sampling Techniques was used for the sampling collection. The field was divided into quadrants or sub units. A simple random sample was taken from each strata or unit. This technique is important because it makes a statement about the sub population, and also increases the accuracy of estimate over the entire population. The samples were properly collected using a soil spade. Every crop residue was scraped off of the soil surface before transfer into the sample containers.

2.1 ANALYSIS ON THE PHYSICAL AND CHEMICAL PEOPERTIES OF THE SAMPLED SOIL: The tested soil was obtained from Delta State University, Campus 3 site, Abraka, Delta state. The basic physical and chemical


properties analysis of the soil, after air drying is shown below, followed by result of analysis in the appendix. PARTICLE SIZE ANALYSIS: Weighed 50g of soil sample, added about 5ml of hydrogen peroxide to remove organic matter, added 50mls Calgon solution, Shaked in the ground shaker for 2hrs. Using 0.05mm sieve washed the sample into a 1 litre-measuring cylinder until the water coming out becoming clear. It is assumed the silt and clay must have passed through the 0.05mm sieve leaving sand fraction. The final volume in the cylinder is noted. It was Shaked vigoriously and 20mls was taken from the colloidal solution at a certain depth, which is determined by calculation. This is the weigh of claying and silt. The sample was Oven dried and the weight was noted. After 2hrs, less or more depending on calculation, another 20mls was taken. It was Oven dried and the weight was noted.

CHLORIDE DETERMINATION: Weighed 5g of air dried soil (passed 2mm sieve), added 25ml of distilled water, mixed with mechanical shaker for 30mins, filtered with the filter paper. Took 10ml of the filtrate into 250ml volumetric flask, added 40ml of distilled water, to the solution in the volume flask, add about 0.5g NaHCO3 and 1ml of 5% K2Cr2O4 solution. Titrated with 0.02N AgNO4 solution, swirling the flask continuously till the first permanent appearance of red orange in the yellow chromate solution.


Determination of Exchangeable Ca, Mg, K, Na, Mn and effective CEC in soil: To 5g of soil samples, 50mls of NH4AC solutions was added and mixed on a mechanical shaker for 2hrs. The clear supernatant was carefully decanted. The exchangeable cations in the A.A.S were

determined. Effective CEC in it was calculated by the sum of exchangeable bases.


pH Determination: Weighed 20g of air-dry soil (passed 2mm sieve) into a 50ml beaker, added 20ml of distill water and allowed to stand for 30mins and stirred occasionally with a glass rod. Inserted the electrode of the pH meter into the partly settled suspension and measured the pH. The suspension was not stirred during measurement. The result was reported as soil pH measured in water. The pH meter was calibrated with pH & and pH 4 buffer solution before use.

2.2 ISOLATION AND ENUMERATION OF BACTERIA IN THE SOIL SAMPLES: Ten (10) gram of each soil samples were dissolved in 9ml sterile distilled water. From the solution, ten-fold serial dilutions in the ranges 10 -1 to 10-5 were prepared. 1ml aliquots of sample dilutions were seeded in sterile petri dishes and total heterotrophic bacterial count was determined by pour plate technique using nutrient agar which can support the growth of non-fastidious bacteria. The Nutrient agar plates were incubated aerobically at 37oC for 24-48 hours. Visible numbers of growth colonies (between 30 and 300) were multiplied by the reciprocal of the dilution factors, and recorded as colonyforming units per gram (cfu/g) of soil.

2.3 SUB-CULTURING: Characteristic colonies on the Nutrient agar plates were picked using a sterile wire loop and streaked on nutrient agar and incubated at 37OC for 24 hours. This process was repeated until pure cultures of


bacterial isolates were obtained. The isolates were maintained on agar slants and stored in the refrigerator at 4oC until required for characterization. 2.4 ISOLATION AND ENRICHMENT OF DDT DEGRADING

BACTERIAL ISOLATES: The enrichment and degradation potential of DDT were conducted in Minimal salt medium containing KNO3 (1.0g), MgSO4.7H20 (1.0), CaCl2.6H20 (0.1g), FeSO4 (0.05g), trace element solution (250ml), phosphate buffer (1M; PH7.0) (20ml). Trace element solution comprised: SnCl2 (0.05g), KI (0.05g), LiCl (0.05g), MnSO4.4H20 (0.08g), HB03 (0.50g), ZnSO4.7H20 (0.10g), CoCl2.6H20 (0.10g), and NiSO4.6H20 (0.10g) BaCl2 (0.05g), Ammonium molybdate (0.05g) and distilled water (1000ml) and DDT about 100ppm as carbon source. The PH was adjusted to 7.0. Cultures were incubated in test tubes containing 9 ml of the mineral salt medium with mouth plugged with sterile cotton wool and incubated at room temperature (25oC) for a period of three weeks. For the bacterial isolation from enrichment culture, transfers to fresh mineral salt medium amended with DDT using about 10% of inoculums from the previous enrichment was done weekly and incubated at 28oC. This procedure was repeated for four successive transfers. Pure cultures were isolated from enrichments by plating out on nutrient agar. Discrete single colonies were selected and inoculated on Minimal agar medium amended with DDT. The process was repeated severally to obtain pure cultures capable of growth on DDT

2.5 DETERMINATION OF GROWTH PROFILE IN DIFFERENT CONCENTRATION OF DDT: The isolates were inoculated into different concentrations of 5ppm, 10ppm, 15ppm, 20ppm DDT minimal salt medium and control (minimal salt medium and the bacterial isolates only). This was done to determine the tolerance level, degradation of DDT, were it serves as carbon source. The cultures were then incubated at ambient temperature for four weeks. The optical density (OD) was determined by measuring the turbidity at 540nm after 30 days using spectrophotometer and pH by Hanna microprocessor pH meter.

2.6 SHAKE FLASK BIODEGRADATIONOF THE DDT BY THE BACTERIAL ISOLATES: The bacterial isolates were screened for the ability to degrade the petroleum compounds present in the DDT samples collected. The ability of the bacterial isolates to utilize the DDT as their sole sources of carbon and energy was determinate using the growth turbidity test according to the methods of Okpokwasili and Okorie (1988). This was carried out by dispensing 100ml of sterilized mineral salt medium into sterile conical flasks (Zajic and Supplison, 1972). In each flask DDT (100 ppm) was added and the flasks were inoculated with 1.0 ml cell suspension of the isolates in sterile mineral salt medium. The mouth of each conical flask was covered with sterile cotton wool. Among the

flask, there was a control which was not inoculated. The flasks were incubated at 37C on a rotary shaker, at a shaking rate of 120 rpm for seven days. At the end of the incubation, the optical density (OD) of each culture was measured at 520nm using Comspec Visible Spectrophotometer. The OD and PH value of each experimental set-up was recorded at time interval of 0, 5, 10, 15, 20, 25 and 30 days.







ISOLATED BACTERIA: The pure bacterial isolates were identified on the basis of their cultural, morphological and biochemical tests. The pure cultures of the bacterial isolates were subjected to various morphological and biochemical characterization tests such as color, shape, elevation, margin , Catalase test, oxidase, citrate, fermentation of sugars, In order to determine the identity of bacteria isolates, results were compared with standard references of Bergeys Manual of Determinative Bacteriology 2nd edition (Buchanan and Gibbon, 1974). 2.8 GRAM STAINING: The gram staining techniques was done on the basis of the component of the cell wall. Organisms which retained the colour of the initial stain are known as gram-positive organisms, while those which do not retain the primary stain when decolorized by gram alcohol are gram negative. The non retention of the stain is due to the cell composition and less lipid activity. The gram staining reagents include: Crystal violet (primary stain),

gram iodide (mordant) ,70% alcohol ( decolouriser ), safranin ( secondary stain ). A drop of sterile distilled water was placed on a clean grease free slide. The inoculating wire loop was flamed until red hot. The loop was allowed to cool and a small portion of the organism to be gram stained was picked and smeared in the drop of water on the slide. The slide was then air dried. It was heat fixed by passing it over flame twice. The smear was stained with 1% crystal violet for 1 minute and washed with distilled water. Gram iodine was added as a mordant for one minute. This was drained off and 75% alcohol was added for 30 seconds. This acts as a decolorizer. The above is termed primary staining. The slide was then rinsed with distilled water. The slide was finally flooded with counter stain, safranin for 1 minute and washed off with distilled water and air dried. The slide was observed under the microscope oil immersion x l00 objective lens. The gram positive organisms appeared purple while the gram negative organisms appeared red.

2.9 BIOCHEMICAL TEST: Catalase Test: This test is used to demonstrate the presence of enzyme catalase, which catalyses the release of oxygen from hydrogen peroxide. The pure culture

of the test organism was placed and added to a drop of 3% hydrogen peroxide solution on a clean slide. The production of gas bubble from the surface indicates positive result.

Oxidase Test: This test helps in identifying the enzyme called oxidase produced by microorganisms. A piece of filter paper was soaked in a few drop of oxidase reagent (Tetramethyl- p- phenylenediamine dichloride). A colony of the test organism was then smeared on the soaked filter paper. An oxidase producing organisms on the filter paper oxidized the phenylenediamine in the reagent to deep purple colour. This change in colour to deep purple within 10 seconds indicates positive result. Coagulase Test: This test is carried out to determine the enzyme coagulase. The test distinguishes pathogenic staphylococcus aureus from other non-pathogenic strains of staphylococcus. A colony of the test organisms was emulsified with sterile normal saline solution on a clean slide using a sterile wire loop. A drop of human plasma was added and mixed with emulsion. The positive coagulase organisms showed clumping while negative coagulase organisms showed no clumping.

Indole Test: This test helps in the identification of enterobacteriae. The test organism was inoculated in a test tube containing 3ml of peptone water and incubated at 370c for 24 hours. About O.5 ml of kovac's reagent which contains

I-p-dimethylaminebenzaldehyde was added. The test tube was shaken gently. The development of rose pink or purple coloration on the surface of the medium indicated a positive reaction of indole production within 10 minutes; while no colour change indicates negative reaction.

Urease Test: This test is used to show if the test organism has the ability to produce the enzyme Urease which catalyze the breakdown of urea to produce ammonia. The medium employed was urea agar base. The sterilized medium was dispensed into bijou bottles. Finally, the test bacterial isolates was inoculated into the medium and incubated at 37OC for 24-48 hours. A change in colour from yellow to red-pink indicated a positive result.

Citrate Utilization Test: Simmon citrate agar is used for this test. Citrate utilization is used to detect organisms that utilize citrate as a carbon and energy source for growth; and ammonium salt as the sole nitrogen source. The medium was made in slants by dispending 5ml of the medium into the bijou bottles and then autoclaved at 1210c for l5 minutes. The slants were inoculated with the test organisms and incubated at 350c for 24 hours. The medium colour change from green to blue indicated a positive result while no change in colour indicated negative result.


Sugar Fermentation Test, A test solution containing 10ml peptone water, 5g of NaCl, 2.5mg of phenol red was prepared in a litre of distilled water. Ten ml of this solution was measured out and 1g of sugar added, thoroughly mixed and autoclaved at 121 0.5oc for 5mins. Sub cultured organisms from the prepared pure culture for 24 hours was inoculated into each of the ten ml solution and incubated at 37oc for 48 hours. Changes in colour from red to yellow indicate positive results.



Representation on table 1 are results of the physiochemical parameters of the various soil samples collected from uncultivated soil. The result from the analysis is showing that the soil used is sandy loam, and is having the pH of 8.00, electrical conductivity of 347.1, total organic carbon of 1.05%, total nitrogen of 0.07%, total phosphorus of 0.003ppm, sodium of 0.954ppm, potassium of 0.777ppm, and calcium of 27.859ppm, very coarse sand (VCS) of 1.64, coarse sand (CS) of 9.48, medium sand (MS) of 29.48, fine sand (FS) of 23.88, and very fine sand (VFS) of 1.27.the soil samples possess a total sand of 66%, total silt of 16% and total clay of 18%, which shows the consistency of the soil and nutrient composition that is favorable to microbial population. Table 2, shows the total heterotrophic isolation of bacteria counts from each of the ten (10) soil samples collected from uncultivated soil. A-J represents each sample of the ten soil samples ranging from the bacteria counts of 1.01105 1.09107. Table 3, shows the growth pattern of isolates in minimal media with DDT. The signs ++++ represent heavy growth of the organisms on the minimal media with DDT, +++ shows moderate growth, ++ shows little growth, + shows low growth while show growth. The organisms + and signs were unable to utilize the DDT.

Table 4 shows the morphological and biochemical characteristics of the bacteria isolates. The organisms isolated from the pure culture were identified on the

basis of their cultural, morphological and biochemical tests. The pure cultures of the bacteria isolates were subjected to various morphological and biochemical characterization test such as colour, shape, elevation, catalase test, oxidation, citrate, fermentation of sugars. The gram staining techniques was done on the basis of the cell wall. Organism which retained the colour of the initial stain is known as gram positive organisms, while those which do not retain the primary stain when decolorized by gram alcohol are gram negative. The characteristics of the organisms isolated are shown by their elevation, margin, colour, shape, opacity and sizes. Most organism has convex as elevation which is a common characteristics among the organisms and other characteristics such as circular, opaque, translucent, large, small, medium, cream, entire, yellow, green, white, and smooth. Table 5, shows the occurrence of the bacteria isolates in the soil sample. Pseudomonas sp. Occurred twice resulting to 20%, Bacillus sp. occurred 3 times resulting to 30% Proteus sp, occurring once resulting to 10%, micrococcus sp. once resulting to 10%, Enterobacter sp. occurred once resulting to 10% and staphylococcus sp. twice results to 20%. Figure 1, shows the graph of the OD (optical density) reading from biodegrading of DDT by microorganism isolates from uncultivated soil. The bacteria in the graph are the isolates that were screened fit for biodegradation and have the ability to degrade the petroleum compounds present in the DDT samples collected. The ability of the bacteria isolate to utilize the DDT as their

sole source of carbon and energy was determined using the growth turbidity test. The bacteria that were able to utilize the DDT are pseudomonas sp., Bacillus sp., and micrococcus sp. Optical density reading was observed to increase steadily until about the 20th day before dropping gradually. Figure 2, shows the graph of the pH reading from biodegradation of DDT by microorganism isolated from uncultivated soil. The pH readings reduced from 7 to about 4.5 from day 0 to 25.



FIELD CODE UNCULTIVATED PH EC (us/cm) % TOC % TOTAL NITROGEN % TOTAL PHOSPHROUS NA (ppm) K (ppm) Ca (ppm) VSC CS MS FS VFS % TOTAL SAND % TOTAL SILT % TOTAL CLAY % TEXTURE 8.00 347.1 1.21 0.10 0.003 0.954 0.777 27.859 1.64 9.48 29.48 23.88 1.27 66 16 18 SANDY LOAM

KEY: EC- Electrical conductivity, TOC- Total organic compounds, PHNegative log. of hydrogen ion, VSC- Very coarse sand, CS- coarse sand, MSMedium sand, FS- Fine sand, VFS- Very fine sand, Na- Sodium, Ca- calcium, K- potassium.




BATERIA COUNT 1.03105 1.08102 1.09107 1.07106 1.09105 1.07104 1.06103 1.04102 1.01105 1.08105




Table 3:



Pseudomonas sp. Proteus sp. Enterobacter sp. Bacillus sp. Staphylococcus sp. Micrococcus sp. Control -

++++ + + +++ + +++

++++ = heavy growth +++ = moderate growth ++ = little growth + = low growth - = growth



Characteristics Gram staining

B1 Gram + cocci in cluster + + + + Convex

Motility Spore stain Biochemical Catalase Urease Indole Oxidase Coagulase Citrate Glucose Lactose Maltose Sucrose Elevation Margin Colour Shape

B2 Gramve rod in single + + + + + + Convex

B3 Gram + rod in single + + + + Convex

B4 Gramve rod in single -

B5 B6 Gram- rod Gram +ve cocci in single + + + + + Swarming Serrated Cream Circular + + + + + Convex Entire Yellow Circular

+ + + + Low convex Entire Entire Smooth Smooth Yellow Green White Cream Circular Circular Circular Circular

B1= Micrococcus sp., B2= Pseudomonas, B3= Bacillus sp., B4= Enterobacter sp., B5=Proteus sp., B6 = Staphylococcus sp. + : Positive reaction, - : Negative reaction






% OF

Bacillus sp. Micrococcus sp. Pseudomonas sp. Enterobacter sp. Staphylococcus sp. Proteus sp. TOTAL

3 1 2 1 2 1 10

30 10 20 10 20 10 100










OD readings for biodegradation of DDT by microorganisms isolated from uncultivated soil

0.4 0.35 0.3 0.25 0.2 0.15 0.1 0.05
Pseudomonas sp Bascillus sp. Micrococcus sp. Mixed culture

) m 0 6 D ( y s n e d l a c i t p O

0 0 5 10 15 Days 20 25 30




pH readings for biodegradation of DDT by microorganisms isolated from uncultivated soil

8 7 6 5 4 3 2 1 0 0 5 10 15 Days 20 25 30
Pseudomonas sp. Bacillus sp. Micrococcus sp. Mixed culture


H p

CHAPTER 4 DISCUSSION OF RESULT In this study, the physiochemical parameters of the sampled soil were examined. The soil was found to be a sandy loam in texture. It contained pH of


8.00, electrical conductivity of 347.1, total organic carbon of 1.05%, total nitrogen of 0.07%, total phosphorus of 0.003ppm, sodium of 0.954ppm, potassium of 0.777ppm, and calcium of 27.859ppm, very coarse sand (VCS) of 1.64, coarse sand (CS) of 9.48, medium sand (MS) of 29.48, fine sand (FS) of 23.88, and very fine sand (VFS) of 1.27.the soil samples possessed a total sand of 66%, total silt of 16% and total clay of 18%, which shows the consistency of the soil and nutrient composition that is favorable to microbial population. Ten soil samples collected from uncultivated soil was analysed. The soil samples labeled A-J, had bacteria counts ranging from 1.01105 -1.09107. cfu/mL. Pure six bacteria isolates were identified from the uncultivated soil sample. The identity of the isolates was determined through cultural, morphological and biochemical characteristics. The six bacteria were Bacillus sp., Micrococcus sp., Proteus sp., Pseudomonas sp., Enterobacter sp. and Staphylococcus sp. The bacteria isolated showed different occurrence level. In the ten samples collected, Bacillus sp. occurred 3 times, Micrococcus sp. occurred once, Enterobacter sp. occurred onces, pseudomonas sp. occurred twice,

Staphylococcus sp. occurred twice and Proteus sp. occurred once. The six isolated bacteria were inoculated into a minimal media with DDT as a sole carbon source. The bacteria showed different growth pattern which indicated their individual ability to utilize and degrade DDT. Signs were used to show their growth rate. In the minimal media with DDT, pseudomonas sp.

showed a more growth ability, it was followed by Bacillus sp. and micrococcus sp. having the same growth rate. Enterobacter sp., proteus sp. and Staphylococcus sp. on the other, had little growth and was weak in utilizing and degrading DDT as a sole carbon source. Optical density (OD) from biodegradation of DDT by the bacteria isolated from uncultivated soil was put in a graph format. The bacteria in the graph were those screened fit to have the ability to utilize and degrade DDT. The growth turbidity test was used to determine the ability of the bacteria isolated to be able to utilize DDT as their sole carbon source. It was observed that the isolated Bacillus sp. were able to degrade the dichlorodiphenyltrichloroethane (DDT) in the minimal medium as its carbon source with maximum growth turbidity of 0.24 and 4.4 for pH of the medium at day 30. However, at higher concentrations, it did not perform well. Bacillus sp. was able to utilize the

dichlorodiphenyltrichloroethane (DDT). The pH reading was 4.4, while the turbidity was 0.24. Pseudomonas sp. was the second best organism that degraded the dichlorodiphenyltrichloroethane (DDT), with pH 4.4 and turbidity 0.22, and micrococcus sp. was the third best organism that degraded the DDT, with pH 4.5 and turbidity 0.18 at 100ppm at day 30. Bacillus sp. isolated from the soil degraded dichlorodiphenyltrichloroethane (DDT), resulting in the lowest pH values 4.4 and also the highest turbidity 0.24. This feature supports the fact that different species or strains of a particular organism responded differently to organic pollutants. From studies, bacteria

belonging to the Bacillus sp., micrococcus sp. and pseudomonas sp. are capable of degrading toxic persistent organic pollutants. From this study Bacillus sp. showed the highest potential in degrading dichlorodiphenyltrichloroethane (DDT) as carbon and energy source. However, degradation by mixed culture of the bacteria isolates was higher than that of any individual isolates. Growth turbidity of DDT degradation is greatly enhanced in a mixed culture perhaps due to their synergistic effect. The result obtained in this study is in agreement with the result of previous studies Alexander (1996), Muller (1998), Katayama (1993), Hect (2004), and Aislabie (1997). The degradation of

dichlorodiphenyltrichloroethane (DDT) was expressed by the increase in turbidity (cell mass) of the degrading bacteria and decrease in pH. The decrease in pH is agreeable because the course of degradation will result in the production of acids and its intermediates, either organic or inorganic depending on what is produced from the degradation pathway, through in some cases they are organic acids with carboxyl groups, and increase in cell mass shows the utilization of carbon (DDT) as the principal energy source. In addition, the increase in the turbidity and decrease of pH of both medium depicts microbial growth, as in the general knowledge of microbial substrate utilization during biodegradation.


CONCLUSION AND RECOMMMENDATION This study showed that there are microorganisms in the tropical soil previously not exposed to DDT that can partially degrade DDT. This study identified three DDT biodegrading bacteria, Pseudomonas sp., micrococcus sp., and bacillus sp., which are ubiquitous in non polluted soils, and are able to remediate soils

polluted with Dichlorodiphenyltrichloroethane (DDT) and other organic pollutants, contaminated by means of improper disposal methods.



The weight of silt = weight of clay and silt weight if clay The final weight of the clay and silt in he total volume =

total volume

the initial weight.

Volume taken (20ml) The sand fraction :- Dry the sand fraction in the oven, pass it through sets of sieves 1mm 0.5mm,0.25, 0.1, 20.1, which is represented as VCS,CS,MS,FS,VFS respectively. Calculation :- % clay = weight of clay Total weight of sample % silt % sand 2 CHLORIDE REACGENTS: Apparatus: 100ml volumetric flask Filter papers Mechanical shaker Reagents: 0.1m AgNO3 (16.987g AgNO3/litre) 5%potassium chromate solution

x 100

=weight of silt Total weight of sample

x 100

= Addition of sand particles in all the sieves DETERMINATION APPARATUS AND

Sodium bicarbonate salt (0.5g/analysis)

3 DETREMINATION OF EXCHANGEABLE Ca, Mg, K, Na, Mn and effective CEC in soil: Apparatus: 100ml volumetric flask Filter paper

Mechanical shaker A. A. S. Reagents Acetic acid glacial and NH4OH conc. Ammonium acetate solution, in PH 7. Add 58ml of glacial acetic acid to about 600ml of distilled water in a 2 litre beaker. Add 70ml conc. NH4OH (s. g 0.9). The NH4OH is best added under a fume hood through a long stemmed glass funnel so that it is introduced into the bottom of the acid solution. Cool the solution and adjust to PH 7.0 with acetic acid or NH4OH using a PH meter.

Transfer the solution into a liter volumetric flask and dilute to volume. Mix it in a Pyrex reagent bottle.


Apparatus: Glass electrode pH meter Reagents 0.01m CaCL2 Distilled water












0 5

10 15





4.9 4.9

4.5 4.7

25 30
4.4 4.4

Pseudomonas sp. 0.05 0.09 0.14 0.19 0.28 0.25 0.22 7.1 6.4 5.8 Bacillus sp. 0.04 0.08 0.15 0.21 0.30 0.27 0.247.0 6.1

5.6 5.2


Micrococcus sp. 0.05 0.07 0.11 0.14 0.16 0.19 0.18 7.1 6.9 6.6 6.1

5.6 5.0

4.5 4.1

Mixed culture 0.06 0.10 0.16 0.23 0.29 0.33 0.37 7.1 6.2 5.3 5.1 4.7 4.4

6 OD


BACTERIA ISOLATES 0ppm Bacillus sp. Micrococcus sp. 0.09 0.14 0.13 OD VALUES (at 600nm) 20ppm 0.17 0.15 65 50ppm 100ppm 0.20 0.18 0.25

Pseudomonas sp. Mixed culture

0.11 0.19

0.17 0.27

0.19 0.27

0.21 0.30

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