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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
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