TITLE PAGE

DEGRADATION OF DICHLORODIPHENYLTRICHLOROETHANE(DDT) BY BACTERIAL

ISOLATES FROM UNCULTIVATED SOIL

BY

NJOETENI KATE AGATHA FOS/SLT/06/07/116757

A PROJECT WORK SUBMITTED TO THE DEPARTMENT OF ENVIRONMENTAL SCIENCE TECHNOLOGY,

FACULTY OF SCIENCE, DELTA STATE UNIVERSITY, ABRAKA.

IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE AWARD OF B.Sc (HONS).

DEGREE IN SCIENCE LABORATORY TECHNOLOGY, (ENVIRONMENTAL SCIENCE TECHNOLOGY OPTION).

JUNE, 2012

1

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 DATE(PROJECT STUDENT)

_____________________ _______________MR. EHWARIEME D. AYO DATE(PROJECT SUPERVISOR)

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

2

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.

3

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.

4

TABLE OF CONTENTS

i. Title page - - - - - - - - i

ii. Certification- - - - - - - - ii

iii. Dedication - - - - - - - - iii

iv. Acknowledgement - - - - - - - iv

v. Abstract - - - - - - - - v

vi. Table of content - - - - - - - vi

Chapter one

1.0 Introduction- - - - - - - - 1

1.1 What is DDT - - - - - - - - - - 2

1.1.1 Origin and history of DDT - - - - - 2

1.1.2 Chemistry of DDT - - - - - - - 3

1.2 Application of DDT - - - - - - 4

1.2.1 Application of DDT to the Environment - - - 4

1.2.2 Application of DDT as an insecticide - - - - 6

1.2.3 Application of DDT for disease control- - - - 8

1.3 Effect of DDT - - - - - - 9

1.3.1 Effect due to Transport of DDT - - - - - 9

1.3.2 Effect due to Metabolism of DDT - - - - 10

1.3.3 DDT effect on health - - - - - - 10

1.3.4 DDT Effects on children - - - - - - 11

5

1.3.5 DDT effect to non laboratory mammal- - - - 13

1.3.6 DDT effect to microorganism - - - - - 13

1.3.7 DDT effect to aquatic invertebrates - - - 13

1.3.8 DDT effect on egg shell- - - - - - - 14

1.4 Fate of DDT to the Environment - - - - - 15

1.5 Biodegradation of DDT - - - - - - 19

1.5.1 Biodegradation of DDT in air - - - - - 20

1.5.2 Biodegradation of DDT in water - - - - - 20

1.5.3 Biodegradation of DDT in sediments and soil - - - - 21

1.6 Current status of DDT - - - - - - - - - - - - - 25

1.7 Aims and Objective- - - - - - 25

1.8 Research problems- - - - - - - - 26

1.9 Scope of the study- - - - - - - - 26

1.10 Limitation of study- - - - - - - 26

Chapter two

2.0 Collection of soil sample - - - - - - 27

2.1 Analysis on the physical and chemical properties of the samples- -28

2.1.1 Particle size analysis - - - - - - 28

2.1.2 Chloride determination - - - - - 28

2.1.3 Determination of exchangeable cations - - - 28

2.1.4 PH determination - - - - - - - 29

2.2 Isolation and Enumeration of bacterial in the soil

6

Samples - - - - - - - - 29

2.3 Sub-culturing - - - - - - - - 29

2.4 Isolation and enrichment of DDT degrading bacterial

Isolates - - - - - - - - 30

2.5 Determination of growth profile in different concentration

Of DDT- - - - - - - - - 31

2.6 Shake-flask biodegradation of the DDT by the bacterial

Isolates - - - - - - - - 31

2.7 Identification and characterization of the isolated

Bacteria - - - - - - - - 32

2.8 Gram staining- - - - - - 32

2.9 Biochemical test - - - - - - - 33

Chapter three

3.0 Results - - - - - - - - - 36

Chapter Four

4.0 Discussion - - - - - - - - 46

4.1 Conclusion - - - - - - - - 49

Appendix - - - - - - - - - 50

References - - - - - - - - 53

ABSTRACT

7

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 1.01×105 -1.09×107 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.

CHAPTER 1

8

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

9

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 Müller, 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

10

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

11

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 non-

living 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

12

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 long-

term 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 1.5–3 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

13

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

14

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

the insecticide while feeding on the plants.

2. 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.

3. 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 protectant (PIPs), are insecticidal substances

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

15

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 near-

worldwide 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

16

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 person’s diet comes

from meat, fish, poultry, and dairy products. Leafy vegetables generally contain

17

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 1995–1996, 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.

18

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 long-

term 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

19

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, children’s

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 8½-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 mother’s 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

20

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)

1.3.5 DDT TOXICITY TO NON LABORATORY MAMMAL:

Experimental work suggests that some species, notably bats, may have been

affected by DDT and its metabolites. Species which show marked seasonal

21

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 µg/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 0.3µg/litre. Toxic effects

include impairment of reproduction and development, cardiovascular

modifications, and neurological changes. Daphnia reproduction is adversely

affected by DDT at 0.5µg/litre. The influence of environmental variables

(such as temperature, water hardness, etc.) is documented but the mechanism

22

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

23

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 1.30–1.75% for

the 49-year-aged soil, but were 4.00–15.2% 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

24

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 (0–1.3 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

5–10 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, 4.0x10-

6, 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,

25

respectively. Laboratory studies of the air/water partition coefficient of DDE

indicate that it will volatilize from seawater 10–20 times faster than from

freshwater (Atlas et al., 1982). Volatilization from moist soil surfaces can be

estimated from the Henry’s 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 4.5–4.8), 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 octanol-

water partition coefficients of 6.91, 6.51, and 6.02, respectively for the p,p’-

isomers 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

26

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

51,000–100,000 in fish, 4,550–690,000 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

27

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

28

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 high-

throughput 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

29

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 C12C–Cl 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

30

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

31

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 1995–1996 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 three-

quarters of the soils having ratios above 1 (Aigner et al., 1998). In a study of

32

forest soils in Maine, the half-life for the disappearance of DDT residues was

noted to be 20–30 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

33

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 1982–1987 and 1989–1993 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

34

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

35

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.

CHAPTER 2

METHODOLOGY

36

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

37

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.

2.1.2 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.

2.1.3 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

38

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

exchangeable bases.

2.1.4 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 colony-

forming 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

39

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

40

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

41

flask, there was a control which was not inoculated. The flasks were incubated

at 37°C 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.

2.7 IDENTIFICATION AND CHARACTERIZATION OF THE

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 Bergey’s 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),

42

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

43

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

44

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.

45

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.

CHAPTER 3

RESULT REPRESENTATION

46

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 1.01×105 -

1.09×107.

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

47

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

48

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.

3.0 PHYSIOCHEMICAL PARAMETERS OF SAMPLED SOIL:

49

FIELD CODE

UNCULTIVATED

PH 8.00

EC (us/cm) 347.1

% TOC 1.21

% TOTAL NITROGEN 0.10

% TOTAL PHOSPHROUS 0.003

NA (ppm) 0.954

K (ppm) 0.777

Ca (ppm) 27.859

VSC 1.64

CS 9.48

MS 29.48

FS 23.88

VFS 1.27

% TOTAL SAND 66

% TOTAL SILT 16

% TOTAL CLAY 18

% TEXTURE SANDY LOAM

KEY: EC- Electrical conductivity, TOC- Total organic compounds, PH-

Negative log. of hydrogen ion, VSC- Very coarse sand, CS- coarse sand, MS-

Medium sand, FS- Fine sand, VFS- Very fine sand, Na- Sodium, Ca- calcium,

K- potassium.

3.1 BACTERIA COUNT IN THE UNCULTIVATED SOIL SAMPLES:

Table 2:

50

SAMPLES BATERIA COUNT

A 1.03×105

B 1.08×102

C 1.09×107

D 1.07×106

E 1.09×105

F 1.07×104

G 1.06×103

H 1.04×102

I 1.01×105

J 1.08×105

3.2 THE GROWTH PATTERN OF ISOLATES IN MINIMAL

MEDIUM WITH DDT:

Table 3:

51

BACTERIA ISOLATES

TUBIDITY

Pseudomonas sp. ++++

Proteus sp. +

Enterobacter sp. +

Bacillus sp. +++

Staphylococcus sp. +

Micrococcus sp. +++

Control -

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

3.3 MORPHOLOGICAL AND BIOCHEMICAL CHARACTERISTICS

OF THE BACTERIA ISOLATES:

Table 4:

Characteristics B1 B2 B3 B4 B5 B6

52

Gram staining Gram + cocci in cluster

Gram-ve rod in single

Gram + rod in single

Gram-ve rod in single

Gram- rod in single

Gram +ve cocci

Motility - + - - + -Spore stain - - + - - -BiochemicalCatalase + + + + + +Urease + + + - + +Indole - - - - - -Oxidase - + - - - -Coagulase - - - - - +Citrate + + - + + +Glucose + + + + + +Lactose - - - + - -Maltose - - - - - -Sucrose - - - - - -Elevation Convex Convex Convex Low

convexSwarming Convex

Margin Entire Entire Smooth Smooth Serrated EntireColour Yellow Green White Cream Cream YellowShape Circular Circular 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

3.4 OCCURANCE OF THE BACTERIA ISOLATES IN THE SOIL

SAMPLE:

TABLE 5:

53

ORGANISM ISOLATED NUMBER OF OCCURANCE % OF OCCURANCE

Bacillus sp. 3 30Micrococcus sp. 1 10 Pseudomonas sp. 2 20Enterobacter sp. 1 10 Staphylococcus sp. 2 20Proteus sp. 1 10

TOTAL 10 100

3.5OD(OPTICAL DENSITY) READINGS FOR BIODEGRADATION

OF DDT BY MICROORGANISMS ISOLATED FROM

UNCULTIVATED SOIL:

54

FIGURE 1: OD READING VS DAYS

3.6 pH READINGS FOR BIODEGRADATION OF DDT BY

MICROORGANISMS ISOLATED FROM UNCULTIVATED SOIL:

55

FIGURE 2: pH READING VS DAYS

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

56

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 1.01×105 -1.09×107.

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.

57

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

58

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.

59

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

60

polluted with Dichlorodiphenyltrichloroethane (DDT) and other organic

pollutants, contaminated by means of improper disposal methods.

APPENDIX

1 PARTICULATE SIZE ANALYSIS CALCULATION: This is the

weight of clay.

The weight of silt = weight of clay and silt – weight if clay

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

61

total volume x 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 x 100

Total weight of sample

% silt =weight of silt x 100

Total weight of sample

% sand = Addition of sand particles in all the sieves

2 CHLORIDE DETERMINATION APPARATUS AND

REACGENTS:

Apparatus:

100ml volumetric flask

Filter papers

Mechanical shaker

Reagents:

0.1m AgNO3 (16.987g AgNO3/litre)

5%potassium chromate solution

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

62

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.

4 pH DETERMINATION APPARATUS AND REAGENT:

Apparatus:

Glass electrode pH meter

Reagents

0.01m CaCL2

Distilled water

63

KLC

5 OD READING FOR BIODEGRADATION OF DDT BY

MICROOGANISMS ISOLATED FROM UNCULTIVATED SOIL:

BACTERIA ISOLATES OD VALUES AT 600nm PH

0 5 10 15 20 25 30 0 5 10 15 20 25 30

Pseudomonas sp. 0.05 0.09 0.14 0.19 0.28 0.25 0.22 7.1 6.4 5.8 5.1 4.9 4.5 4.4

Bacillus sp. 0.04 0.08 0.15 0.21 0.30 0.27 0.247.0 6.1 5.6 5.2 4.9 4.7 4.4

64

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

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 4.1

6 OD VALUES AT DIFFERENT CONCENTRATION OF DDT

AFTER 30 DAYS:

BACTERIA ISOLATES OD VALUES (at 600nm)

0ppm 20ppm 50ppm 100ppm

Bacillus sp. 0.14 0.17 0.20 0.25

65

Micrococcus sp. 0.09 0.13 0.15 0.18

Pseudomonas sp. 0.11 0.17 0.19 0.21

Mixed culture 0.19 0.27 0.27 0.30

REFERENCE

Adeshina, F., 1991. Exposure assessment of chlorinated pesticidesin the environment. J Environ Sci. Health A26(1):139-154.

Adlercreutz, H., 1995. phytoestrogens: epidemiology and a possible role in cancer protection. Envrion. Health Perspect 103(Suppl 7):103-112.

Agarwal, N., sanyal S., khuler G., et al. 1978. Effect of acute administration of Dichlordiphenyl. trichloroethane on certain enzymes of Rhesus monkey. Indian J. Med Res 68:1001-1006.

66

Aiay, S., Owen, P., 2004. Applied Bioreme diatom and Plytoremedation. Springer Verily, Berlin, Heidelberg. pp 36-37.

Aidlejie, J., Davision, A., Frezmern, P., Jardire, D., Karuso, P. 1998. Co. metabolism of Det by an Aerobic Bacterium. In 14 th Austrlerian biotechnology conference Adelaide Australia 113CEH.

Aislabie, JM.,, Richard, NK., Boul, HL., 1997. Microbial degradation of DDT and its Resides. A review N Z J Agric Res 40:269-282.

Alborne, ES., Eglinton, G., Evans, NC et al., 1972. Fate of DDT in seven estuary sediments. Environ. Sci. Technol., 6:914-919.

Alexander, M., 1995. How Toxic Chemical in Soil? Eviron. Sci. Technol., 28(11):2713-2717.

Alexander, M., 1997. Sequestration and bioavailability of Organic Compound in Soil in Linz, DG., Nalkles, DU., Environmentally acceptable Endpoints in Soil. Annapolis, MD: American Academy of environmental engineers, 34-136.

Andrea, M., tomita, R., Iuchini, L., et al., 1994. labouratory studies on violation and mierlization of DDT in soil, reslase of bound residues and description from solid surface. Envrion. Sc.i health., b29 (1): 133-139.

Arisdory, M., 1998. Biodegradation of chlorinated organic compounds by white not fungi. Environ. contain. Toxico., 60;872-276.

Atlas, E., foster, R., Glam, C., 1982. Air sea exchange of high moleculer weight organic pollutants; labouratory studies. Environ. Sci. technol, 16; 283-286.

Baker, RD., Applegate, HG., 1970. Effect of temperature and ultraviolet radition on the persisitence of methyl Parathion and DDT in Soil. Agron J., 62:509-512.

Bevenue, A., 1976. The bioconcentration aspects of DDT in the environment. Residue Rev 61:37-112.

Bidleman, T., 1998. Atmospheric processes, Wet and Dry deposition of organic compound controlled by their Vapor-particle partitioning. Eviron. Sci. Technol., 22(4): 361-367.

67

Bitman, J., lillie, RJ., Cecil, AC., Fries, GF., 1971. Effect of DDT on Reproductive performance of Ceged leghorms. Poultry science, 50:657-659.

Boul, H., 1996. Effects of soil moisture on the fate of radiolabel led DDT and DDE in Vitro. Chemosphere 32(5);855-866.

Boul, HL., Garnhrm, ML., Hucker, D., et al., 1994. Influence of agricultural practices on the levels of DDT and its residuies in soil. Environ. Sci. technol., 28(8): 61-66.

Bouwman, H., Becker, PJ., Coopan, RM., et al 1992. Transfer of DDT used in malaria control to infants via breast milk. Bull World Health Organ 70(2):241-250.

Coulston, F., 1973. Shell Thinning in avian eggs by environmental Pollutants. Eviron. Pollut. 4: 85-152. Intake Regul toxicol Pharmacol 5: 332-283.

Cousins, IT., Mclachlan, MS., Jones KC., 1998. Lack of An Aging Effects on the soil Siphengl Ioring of Polychlorirtaes Siphengls. Environ. Sci. Technol., 32: 2734-2740.

Devise, RP., Dobbs, AJ., 1984. The Prediction of bioconcentration in fish water. Res., 18(10): 1253-1262.

Diamond, J., Owen, R., 1996. Long Term Residue of DDT compound in forest Soil in Marine. Environ. poll., 92;227-230.

Dileep, K., 2008. Biodegradation and Bioremediation Pesticides in soil Concept. Method and Recent Development. Ind. Microbiol., 48: 35-40.

Evans, MS., Noguchi, GE., Rice, CP. 1991. The Biomagnifications of polychlorinated Biphenlyls toxipene and DDT compound in a Lake michgen offshore food Web. Arch environs. contain toxically, 20: 87-93.

Fishbein, L., et al., 1973. DDT and its metabolies polychlorinayed Biphenyls, Chlorodioxins, polycyclic aromatic hydrocarbons, haloethers. Photosentized reductive edchlorination of Chloroaromatic Pesticides. Chemposhere, 30(9): 1655-1669.

68

Fold, UM., Hill, EP., 1991. Organization pesticides in soil sediments and aquatic animals in the upper steel bayous water shed of Mississippi. Arch. Environ. Contem., 20;161-167.

Fries, s., Cherles, G., Evans, WC., 1969. Anaerobic degradation of aromatic compound. Ann. Rev. Microbial 42: 289-317.

Fuhremann, TW., Lictenstein, EP., 1980. A comparative study of the persistency movement and metabolism of six carbon, 14 insecticides in soil and plants. J. agric. food chem. 28:446-452.

Geyer H, sheena P, kotizas D, et al 1982. Prediction of ecotixiocological behaviour of chemicals: relationship between physico chemical properties and bioaccumaltion of organic chemicals in the mussel mythilus edulis chemosphere 11(11): 1221-1134.

Giesy JP vergbrugge DA, Othout RA, et al: 1994 contaminants in fishes from the great lakeinfluenced sections and above dams of three Michigan Rivers I: concentration pf argano chlorine insecticides, polychlorointed biphenyls, dioxin equivalents and mercury. Arch Environ Contain toxical 27:202-212.

Giesy, JP., Verbrugge, DA., Othout, RA., et al., 1994. Conterminant in fishers from the Great Lakes Influenced Sections above dams of the three michigam river. Arch. Environ. Contem. toxicol., 27:202-212.

Gray, N., Moser, G., Moser, L., 1999. Compost decontermination of soil conterminated with chlorinated toxicant. Google patents 5: 902-744.

Harmer, T., 1997. Organochorine contamination of the Canada article and speculation of future trends. Int. J. Envron. Pollut., 8 (½) 51-73.

Hect, N., Guerzi, U., Beard, W., 2004. Anaerobic Conversion OF DDT TO DDX and aerobic stability of DDT in soil. Society of American proceedings, soil science 32:522-527.

Hickey, WJ., 1999. Transformation and fate of Polychorireted Bipherylo in Soil Frankenburger WT, JR, SINs RC. Bioremediation of Contaminated soils. Agron Nonograph 37: 213-237.

Hitch, RK., Day, HR., 1992. Unusual persistence of DDT in some western USA soils. Bull environ. contain. Toxicol., 48:259-264.

69

Hong, Q., Zhing, Hoong, V., 2007. A Microcosm Study on Biromediction of Fenitrotheion Contaminated soil using burkholderic sp. FDS-1. Biorenied. Biograd., 59: 55-61.

Howard, P., Meylan, BL., 1997. Handbook of physical properties of organic chemicals. Boca Ration, FL: CRC press, Lewis Publishers, 3, 18, 49, 518, 723.

Jackques, R., Okeke, B., et al., 2008. Microbial Consortium Bio Augmentation of a polycyclic aromatic hydrocarbons cotenants soil. Bioresearch techno., 99: 22637-2643.

Johnson, G., Jalal, S., 1973. metabolism in micorbail Systems. Residue Rev 61:1-28.

Julius, A., Naidu, R., 2000. Enrichment and Isolation of Non specific aromatic degreds from unique uncontaminated (plant and feacal materials) sources and contaminated soils. Appl Microbio,l 8a: 642-650.

Katayama, Richard, NK., Boul, HI., 1993. Microbioal degradation of DDT and its residues. A review N.Z.J Agric. Res., 40:269-282.

Kumer, M., Philip, L., 2006. Bioremediation of endosultion contaminated soil and water optimization of operation conditions in Laboratory scale reactions. J. hazardous mater, 136: 354-364.

Kurihere, N., Ikemoto, Y., clerk, G., 1998. Dehydrochlorination mechanism of DDT Arelogo Catalyzed by housefly enzymes. Agric, Biol. chem., 52 1831-1833.

Lai, R., saxenxa, D., 1991. Accumnluation metabolism and effect of organichlorine insectides on microorganism. Microdiol Rev, 46: 95- 127.

Leblanc, G., 1995. Trophic level difference in the biocencentraion of chemicals implication in assessing environment biomagnifications. Environ. Sci. technol., 29:154-160.

Lichtenstein, E., Schulz, K., 1960. Translocation of some chlorinated hydrocarbon insecticides into the aerial parts of pea Plants. Agric. food. chem.., 8(6):4520456.

70

Lundholm, C., 1997. Effect of p,p DDE on the calcium and protagaladain metabolism of the eggshell galad, Comparative biochemistry and physiology part C. Pharmacology, toxicology and endiocrinlology, 118(2): 113-128.

Menzie, C., 1980. Treaction types in the environment in Hutzinger O. Reactions and processes New York NY: springer-Verlag Berlin Heildberg, 247-302.

Metcalf, RL., Akpoor, IP., et al., 1973. Model ecosystem studies of the environment fate of six organochlorine pesticides. Environ. health precept 4: 35-44.

Meylan, w., Howard, P., Boethling, R., 1992. Molecular topography/fragment contribution method for predicting soil sorption coefficient methods. Evrions. Sci. technical 28 (8): 1560-1567.

Meylan, W., Howard, PH., 1993. Organic compound with hydrolxl radicals and ozone. Chemosphere 26(2):2293-2299.

Miller, G., Zepp, R., 1979. photo activity of aquatic pollutant absorbed on suspended sediments. Eviron. Sci. Toxicol. 13(7):860-863.

Morrison, DE., Robertson, BK., Alexandra, M., 1999. Possible use of a solid phase extractant to predict availability of DDT, DDE and DDD in Soil. Soil Science,49: 1039-1044.

Nash, R., Beall, L., 1970. Chlorinated hydrobarbon insecticides: root uptake versus vapour contamination of soybeans foliage. science. 168;1109-1111.

Oliver, B., Nilmi, A., 1985. Bioconcentraion factors of some halogenated organic fro rainbow trout: limitation in their use for prediction of environmental residues. Environs. Sci. Techno. 19:842-849.

Pfaender, F., Alexander, 1976. Extensive microbial degradation of DDT in vitro and DDT metabolism by natural communities. J. Agric Food chem. 20: 282-846

Quensen, J., Muller, S., Jain, M., et al., 1998. Reductive dechlonation of DDT to DDMU in Marine Sediment. Microcosms science 280: 722-724.

71

Rack, K., Sidmore, M., Hamlition, D., et al., 1997. Pesticides are in tropical soils. Pure Appl. Chem 69(6)1349-1371.

Reish, DJ., Kauwling, TJ., et al. 1990. Marine and estuarine pollution. J water pollut control Fed., 1424-1462.

Remesh, C., Atur, K., Ajay, Owen, P., 2004. Bioreme Diction Springer Verky Berlin, Heidelbery, pp. 35-37

Robertson, BK., Alexander, M., 1998. Sequestion of DDT and dividing in soil, disappearance of acute toxity but not the compound. Envron. toxically Chem. 17(6): 1034-1038.

Sablyic, A., 1984. Prediction of the nature and strength of soil sorption of organic pollutants by molecular topology. J. agric. Food Chem., 32: 243-246.

Samuel, T., Pillai, MK., 1990. Effect of temperature and solar daiotion on volatilasation, mineralization and degradation of DDT in soil. Eviron. Pollut., 57:63-77.

Sandju, S., Warren, W., Nelson, P., 1978. Pesticides residues in rural potable water. J. on water works assoc. 41-45.

Samuel, T., Pillai, MK., 1990. Effect of temperature and solar redaition on volaisetion, mineralisation and degredation of DDT in soil. Environ, pollut., 57: 63-77.

Schwack, W., 1988. Photo induced additions of pesticide to bio-molecule. J. agric. food chem. 36:645-648.

Sjoeib, F., Anwar, E., Tunggul, O., 1994, laboratory studies of dissipation of DDT from soil and plywood surface. J Envron. Sci. health., B 29 (1): 153-159.

Speicer, BF., Singh, G., Taylor, CD., et al 1996. DDT persistence and volatility as affected by management practices after 23 years. J. Environ. Qual 25: 815-821.

Stanley, C., Barney, J., Heklton M., et al., 1971. Measurement of atmospheric levels of pesticides. Eviron. Sci. technol., 5(5): 430-435.

72

Stewart, D., Chrsiton, D., 1971. longer term persistence of BHC, DDT and chlordane in sandy loam soil. J. soil Sci., 51:379-383.

Swann. EH., Mccell, PJ., Laskowski, DA., et al., 1981. Estimation chemicals by high performance liquid chromatography. Spec. Tech. pub., 737: 43-48.

Thomas, RG., 1990. Volatilization from soil in; Lyman WJ, Rechl UF, Rosenblatt DH, eds hardbook of chemical property estimation method environmental behaviour of orgaic compounds Washingtons, DC: American Chemical society, 16-25 to 16-28.

Tucker, RK., Haegele, HA., 1971. Other Pollutant may cause Thin egg shells.Utah science, pp 47-49.

Tursov, Valdamir, et al., 2002. Dichlorodiphentrichchlorethane (DDT). Unbiquity persisitence and risk environment health perspectives 110(2) 125-128).

Van Eden, HF., Peall, DB., 1996. Beyond slient spring champ men and hall London 322 pp

Veith, G., Defoe, D., Bergstedt, B., 1979. Measuring and Estimating the Bioconcentration Factor of chemicals in Fish. J Fish Res Board Cen 36: 1040-1048.

Verma, A., pillai, MK., 1999. Bioavailability of soil bound residues of DDT and HCH to certain plant. Soil Biol. Biochem., 23 (4): 347-35.

Verna, A., pillia, MK., 1991. Bioavailability of soil bound residue of DDT and HCH to Earth worm. Curr Sci., 61: (120:840-843.

Vollner, L., Klotz, D., 1994. Behaviour of DDT under laboratory and Outdoor conditions in Germany. J. Environ. sci. health 29(1); 161-167.

Woodwell, G., Craig, P., Johnson, H., 1971. DDT in the Biospher: where Does it go? Science 174:1101-1107.

You, G., Salyes, H., Kupferle, et al., 1995. Anaerobic Biomediction of DDT contaminated soil with Non ironic surfactant. Appl. Envrion. Microbio., l3:137-144.

73

Aigner, E., leone, A., 1995. Concentrations And entiometric ratio of Organochlorine pesticides in soils from US. Corbel Eviron Sci Technol 32:1162-1168.

Zepp, GR., et al 1997. Phototchemical transformation of ddt and methoxoychlor degradation products, DDE and DMDE, by sunlight. Arch. Eviron. contam. tosicol. 6:305-314.

74