2.1 Review of literature
Agriculture has been the main occupation in many countries. In India,
more than 70% of the Indian population depends on agriculture (FAO, 2003).
Over the decades, farmers have switched over to commercial farming where
scientific pest management using several anthropogenic pesticides has played
very important role. Today, contribution of pesticides is well recognised and
appreciated in agricultural programmes. This has led to an increased food grain
production that has shown a big leap from 52 million tonnes in 1951- 52 to 100
million tonnes in 1998- 99 (Rai foundation, 2008). The population of India has
already crossed 1 million mark. Food requirements will be of the order of 300
million tonnes with respect to the 143 million ha cultivated area in the country
(Rai foundation, 2008). Hence, the use of agrochemicals like fertilisers and
pesticides has increased, leading to their accumulation in different components
of the environment. Although, pesticides have greatly increased agricultural
production and saved millions of lives from insect borne diseases, the use of
certain pesticides has resulted in the pollution of the environment.
Persistent organic pollutants (POPs) are toxic chemicals that adversely
affect human health and the environment. These can be transported by wind
and water, persist in the environment for long period and get accumulated in the
food chain (Fig. 2.1). The Stockholm convention in 2001 identified twelve
chemicals as POPs. These chemicals, called the “dirty dozen”, comprise of
aldrin, chlordane, 1,1,1- trichloro- 2,2 bis(4- chlorophenyl)ethane (DDT),
dieldrin, endrin, dioxins, heptachlor, hexachlorocyclohexane, mirex, toxaphene,
PCBs and furan (USEPA, 2002).
In modern agricultural practices, DDT is one of the major insecticides
used mostly for cotton, soyabean, peanuts, tobacco and potato crops (Cocco et.
al., 1997). Though first synthesized by Othmar Zeilder in 1874, the insecticidal
properties of DDT were first discovered by the Swiss scientist Paul Hermann
M ller in 1939. M ller was awarded the 1948 Nobel Prize in Physiology and
Medicine for his discovery of the high efficacy of DDT as a contact poison in
several arthropods (Ra and Shampo, 1980). It was used extensively during
8
World War II by allied troops in Europe and the Pacific as well as certain civilian
populations to control insect typhus and malaria vectors. In 1940s, DDT had a
decisive role in eradication of malaria from Europe and United States (Dash et.
al., 2007). When DDT was introduced in 1940s, then Prime Minister of United
Kingdom, Rt. Hon. Winston Churchill in his broadcast on 28th September 1944,
stated "we have discovered many preventives against the tropical diseases and
often against the onslaught of insects of all kinds, from lice to mosquitoes and
back again. The excellent DDT powder, which has been fully experimented with
and is found to yield astonishing results will henceforth be used on a great scale
by the British forces in Burma, and the American and Australian forces in the
Pacific and in India in all theaters…". Thus, in a short span of time, DDT
achieved the unique distinction of saving millions of lives by preventing disease
outbreaks than any other anthropogenic chemical.
Fig. 2.1: Biomagnification of POPs
9
In 1962, the publishing of Silent Spring by Rachel Carson, an American
biologist, catalogued the environmental impacts of the indiscriminate spraying of
pesticides- notably DDT. The book argued that the pesticides, especially DDT,
were poisoning both wildlife and the environment and also endangering the
human health. It was suspected to be a carcinogen. Public reaction to Silent
Spring launched the modern environmental movement in the United States, and
DDT became a prime target of the growing anti- chemical and anti- pesticide
movement during the 1960s. The pro- and anti- DDT arguments finally lead to
the ban of DDT in the U. S. in the 1970s (ATSDR, Atlanta, 1994). But still DDT
was allowed for the health related emergencies due to outbreak of certain
vector borne diseases in social and preventive medical practices (ATSDR,
Atlanta, 1994). The developing world continued the use of DDT in two major
areas of agriculture and public health programmes. The main reason for its vast
usage was its broad spectrum of action on wide variety of pests and the low
cost compared to the other existing pesticides (Walker, 2000). The higher cost
of alternative insecticides is often cited as a reason to continue using DDT
(Attaran et. al., 2000; Curtis and Lines, 2000; Roberts et. al., 2000).
The most conspicuous ecological concern of the DDT impact was on
decline of bird populations of peregrine falcons, bald eagles and ospreys
through inconclusive scientific evidence. Royal Society for protection of birds in
1998 stated that egg shell thinning of some bird species had begun 50 years
before the introduction of DDT (Bailey, 2007). The decline in the population of
birds was the foremost anti- DDT argument. Finally, in 2001, the Stockholm
Convention addressed the global concern of POP pollution in the environment.
A treaty was signed by 98 countries which agreed to reduce or eliminate the
production, use and/ or release of 12 key POPs (USEPA, 2001). The treaty,
which came into effect from 17th May, 2004 outlawed several POPs and
restricted the use of DDT to vector control. The public health use of DDT was
exempted from the ban until any alternatives to DDT were developed. The
treaty was endorsed by most environmental groups. The Malaria Foundation
International (2006) stated: “the outcome of the treaty is arguably better than
10
the status quo going into the negotiations over two years ago. For the first time,
there is now an insecticide which is restricted to vector control only, meaning
that the selection of resistant mosquitoes will be slower than before”.
According to UN estimate, one child for every 30 sec and a million
people per annum died of malaria, mainly in the Sub Saharan Africa. The
WHO/UNICEF Roll Back Malaria (RBM) initiative to reduce 50% of deaths by
2010 in Africa involved use of pyrethroid treated nets, pyrethroid IRS and
artemisinin. But a review of RBM surprisingly showed increase in malaria
deaths in Africa during 1999- 2003 (Yamey, 2004). Thus, the program was a
failure, as feared by some scientists. This led the WHO to reassess its policy on
the basis of all known scientific for and against the use of DDT. Finally, in
September 2006, almost 30 years after it phased out widespread indoor
spraying of DDT, the WHO gave a clean bill to use DDT to combat malaria in
Africa and other areas where vectors are still susceptible to DDT (Dash et. al.,
2007). Thus, with the reintroduction of DDT, it was clear that no other effective
alternative to DDT for controlling disease transmitting vectors. One insecticide
company quoted on its website: “DDT is still one of the first and most commonly
used insecticides for residual spraying, because of its low cost, high
effectiveness, persistence and relative safety to humans. In the past several
years, we supplied DDT 75% WDP to Madagascar, Ethiopia, Eritrea, Sudan,
South Africa, Namibia, Solomon Island, Papua New Guinea, Algeria, Thailand
and Myanmar for Malaria Control project, and won a good reputation from WHO
and relevant countries’ government” (Yorkool Chemicals, 2006).
2.2 Indian scenario
India is one of the foremost countries among the third world to start large
scale use of pesticides for the control of insect pests of public health and
agricultural importance. A total of 145 pesticides are registered for use in India
and production has increased to approximately 85, 000 tonnes (Gupta, 2004).
Pesticide use in India is increasing at the rate of 2- 5% annum. Of the total
amount of pesticides used in India, insecticides contribute to 76% whereas
fungicides and herbicides contribute to 13% and 10% respectively (Mathur,
11
1999). The consumption of pesticides in India has decreased in the long run.
This may be due to the low dosage requirements of many new pesticides and a
gradual shift to the integrated pest management approach. However, the
persistent organochlorines, whose use has been restricted in the western
countries more than a decade ago, are still being used in large amounts in
India. The consumption of pesticides in India is low (0.5 kg/ha) against 6.60 and
12.0 kg/ha in Korea and Japan, respectively (Gupta, 2004). Despite this fact,
there is a widespread contamination of the food stuffs in India. This may be due
to the non- judicious use of pesticides. In India, 51% of the food commodities
are contaminated with pesticide residues and out of these, 20% have pesticide
residues above the maximum residue level (Gupta, 2004). Kumari et. al. (2007)
reported the presence of DDT in rain water from Hisar. John et. al. (2001)
reported the presence of DDT and its metabolites in milk samples collected
from Jaipur city, Rajasthan.
In India, the use of DDT for agricultural purposes was banned in 1989
with a mandate to use a maximum of 10, 000 tons of DDT per annum for the
control of malaria and Kala- azar (Dash et. al., 2007).
A survey conducted by Greenpeace in 1999 indicated severe
contamination of DDT and other persistent organic pollutants in nearby creeks
of Hindustan Insecticides Ltd. factory, Kerala.
Table 2.1: Pesticides detected in few rivers in India (Gupta, 2001)
Source
Cauvery river,
Pesticide
Detected quantity
BHC
> 1000ppb
Methyl parathion
1300ppb
DDT
21.8ppm
Karnataka
Cauvery river,
Karnataka
Yamuna river, Delhi
12
Table 2.2 DDT Concentrations in man (Gupta, 2001)
DDT accumulated in an Indian
12.8-31.0ppm
Average daily intake of DDT in India
238.1-224.1µg/person
Average daily intake of DDT in
20.0 µg/person
Australia
Average daily intake of DDT in
10.8 µg/person
Canada
Table 2.3: DDT Concentration in Few Food Articles in India (Gupta, 2001)
Food article
DDT concentration
Wheat
1.6-17.4 ppm
Rice
0.8-16.4 ppm
Pulses
2.9-16.9 ppm
Groundnut
3.0-19.1 ppm
Potatoes
Bottled milk(Mumbai)
68.5 ppm
4.8-6.3 ppm
Milk (Mumbai, vendors)
97.0 ppm
Butter
3.6 ppm
2.3 DDT, the "atomic bomb"
1,1,1- trichloro- 2,2 bis(4- chlorophenyl)ethane (DDT)
was
first
synthesized in 1874 by German chemist Othmar Zeidler by the reaction of
chloral (C2HCl3O) with monochlorobenzene (C6H5Cl3) in the presence of
concentrated sulphuric acid as a condensing agent (Fig.2.2). 225 parts of
chlorobenzene were mixed with 147 parts of chloral or the corresponding
amount of chloral hydrate and then 1000 parts sulphuric acid monohydrate were
added. Whilst stirring well, the temperature rose to 60oC, and then sinking
slowly to room temperature, the mass then contained solid portions. It was
poured into a large excess of water whereupon the product separated in solid
13
form. It was well washed and could be crystallized from ethyl alcohol, forming
fine white crystals having a weak fruity odour (West and Campbell, 1950).
Fig. 2.2: Synthesis of DDT as done by Zeilder
But it was only in 1939 that Dr. Paul Müller found that DDT quickly killed
flies, aphids, mosquitoes, walking sticks and Colorado potato beetles. Müller
and the Geigy corporation patented DDT in Switzerland (1940), England (1942)
and U.S. (1943).
The first large-scale use of DDT occurred in 1943 when 500 gallons of
DDT were produced by Merck & Company and delivered to Italy to help squelch
a rapidly spreading epidemic of louse-borne typhus. Later in 1943, the U.S.
Army issued small tin boxes of 10 percent DDT dust to its soldiers around the
world who used it to kill body lice, head lice and crab lice.
Some
of
agricultural
pests
controlled
by
DDT
are
various potato beetles, coddling moth (which attacks apples), corn earworm,
cotton bollworm, aphids and thrips of cotton, cotton leaf roller, red cotton bug,
tobacco budworms, pod-borer and pod- catterpillar of pulses, stem borer, top
shoot borer and leaf hopper of sugarcane, lemon butterfly of citrus plants, shoot
and fruit borer of vegetables, aphids of groundnut and thrips of chilli.
14
2.4 Structure
DDT's chemical formula is C14H9Cl5. So, for every molecule of DDT,
there are 14 carbon atoms, 9 hydrogen atoms, and 5 chlorine atoms (Fig. 2.3).
Grummitt et. al. (1945) confirmed the position of Cl- atoms in DDT by refluxing
with alcoholic potash to give the corresponding dichloroethylene compound and
oxidizing the latter with chromic acid to p,p'
- dichlorobenzophenone.
Fig. 2.3: Structure of DDT
A- Skeletral Structure
B- 3D structure
2.5 Properties
In its pure form, DDT is a white, crystalline powder with little odour.
Form
Colourless crystals, technical- waxy solid
Molecular weight
354.5
Vapour pressure
0.025 mPa (109 0C)
Boiling point
185- 187 0C/ 0.05 mmHg
Melting point
108.5-109 0C
Density
0.98-0.99
15
2.6 Solubility
DDT is practically insoluble in water, readily soluble in most organic
solvents. Tables 2.4 and 2.5 describe the solubility in various solvents at
different temperatures (Gunther, 1945; Jones et. al., 1946).
Table 2.4: Solubility of DDT at different temperatures in different solvents
Weight % solubility at
Solvent
0 0C
7.2 0C
24.0 0C
45.0 0C
48.0 0C
Acetone
21.2
27.3
40.3
-
59.0
Benzene
6.8
27.1
44.0
-
57.8
Carbon tetrachloride
9.0
10.5
18.0
34.8
-
Chloroform
18.2
21.9
31.0
47.4
-
Dioxane
8.0
29.0
46.0
-
61.0
Ether
15.0
18.9
27.5
-
-
Ethanol (95%)
0.8
1.0
2.2
-
3.9
1.7
2.4
4.8
-
-
21.0
36.0
51.0
-
62.0
Petroleum ether
(30- 600)
Pyridine
Source: Bidlan, 2003.
Table 2.5: Solubility of DDT in various solvents at 27 0C - 30 0C
Solubility of DDT
S. No.
Solvent
Per 100 ml of
Per 100g of
solvent (g)
solvent (g)
1
Acetone
58
74
2
Acetophenone
67
65
3
Anisole
70
70
4
Benzene
78
89
5
Benzyl acetate
45
43
16
6
Benzyl alcohol
12
11
7
Benzyl ether
41
39
8
Carbon tetrachloride
45
28
9
p- chloroacetophenone
39
33
10
Chlorobenzene
74
67
11
Chloroform
31
-
12
Cyclohexane
15
19
13
Cyclohexanol
10
11
14
Cyclohexanone
116
122
15
o- dichlorobenzene
59
45
16
1,4- dioxane
92
89
17
Ethyl alcohol (95%)
~2
~2
18
Ethyl benzoate
37
54
19
Ethylene dichloride
59
47
20
Ethyl ether
~28
~39
21
Methylene chloride
88
66
22
Propionic acid
16
16
23
Tetrachloroethane
61
38
24
Tetrachloroethylene
38
23
25
Triacetine
10
9
26
1,2,4- trichlorobenzene
44
28
27
1,1,1-trichloroethane
~52
~39
28
Trichloroethylene
64
44
29
o-xylene
57
66
Aliphatic Petroleum Fractions
30
Gasoline
10
13
17
31
Kerosene
8- 10
10- 12
32
Fuel oil 1
8- 11
10- 14
33
Fuel oil 2
7- 10
8- 12
34
Lubricating oil
5
6
35
Refined fly's spray-base
4
5
4- 6
5- 7
kerosene
36
Transformer oil
Coal Tar Fractions
37
Xylene 100
53
61
38
Solvent naphtha
52
60
(Industrial Xylene)
39
Wire Enamel Solvent
60
64
40
Neutral oil
67
66
41
Special hydrocarbon oil
58
53
Pine Distillation Products
42
Pine oil
10
11
43
Turpentines, Spirits
17
20
9
10
Miscellaneous
44
Alox 152 (Methyl esters
of oxidized petroleum
products)
45
Alox 800
18
19
46
Aroclor 1242
48
35
(Chlorinated biphenyls)
47
Aroclor 1248
~30
~21
48
Aroclor 1254
12
8
49
Castor oil
7
7
18
50
Cotton- seed oil
11
12
51
Sesame oil
8
9
52
Linseed oil (raw)
11
12
53
Peanut oil
11
12
54
Iso- propyl cresols
7
7
55
Triton X 100
12
11
Source: Bidlan, 2003.
2.7 Stability
Domenjoz (1944) reported that little or no decomposition occurred on
heating DDT at 150 0C for 24 hours. West and Campbell (1950) heated DDT
very slowly in a boiling tube immersed in a glycerine bath, maintaining at 115
0
C, 120 0C, 125 0C, 130 0C, 140 0C, 145 0C for about one minute each with fairly
rapid current of air passing through. Temperature at which a definite
opalescence first appeared in the silver nitrate solution was 140 0C-145 0C. It
undergoes dehydrochlorination in alkaline solution and at temperatures above
the
melting
point
to
the
non-
insecticidal
1,1-
dichloro-
2,2
bis(4-
chlorophenyl)ethylene (DDE). DDT is generally stable to oxidation.
2.8 Chemical composition of technical DDT
According to Haller and co- workers (1945), theoretically, there are 45
possible dichlorodiphenyltrichloroethanes. Table 2.6 gives the composition of
technical DDT.
19
Table 2.6: Composition of dichlorodiphenyltrichloroethane
Compound
Sample 1
Sample 2
p,p -DDT
66.7
70.5
72.9
63.5
64.5
o,p - DDT
19.0
7.0
Sample 3
72.7
Sample 4
-
76.7
11.9
74.8
15.3
20.9
p,p - DDD
0.3
4.0
0.17
-
o,p - DDD
-
-
0.044
-
1-p-chlorophenyl-2-
0.2
-
-
-
-
-
0.044
-
0.6
0.1
0.034
-
-
0.01
0.006
-
-
0.007
-
-
Chlorobenzene
-
-
-
2.44
p-dichlorobenzene
-
-
-
0.73
Sodium-p-
0.02
-
-
-
-
-
0.005
-
Inorganic
0.1
0.04
0.01
-
Unidentified and losses
6.5
5.1
10.6
19.4
trichloroethanol
2-trichloro-1-ochlorophenylethyl-pchlorobenzene sulphonate
Bis (p-chlorophenyl)
sulphone
α-chloro-α-chlorophenyl
acetamide
α-chloro-α-o-chlorophenyl
acetamide
chlorobenzenesulphonate
Ammonium pchlorobenzenesulphonate
Source: Bidlan, 2003.
20
2.9 Trade and other names
DDT was commercially marketed by many trade names but many of
these names are no longer used since the use of DDT has been discontinued in
many countries. Few of them have been mentioned below (Wasserman et al.,
1982; ATSDR, Atlanta, 1994).
1. Anofex
11. Didimac
21. Kopsol
2. Agriton
12. Digmar
22. Micro DDT 75
3. Arkotine
13. ENT 1506
23. Mutoxin
4. Cesarex
14. Genitox
24. Neocid
5. Chlorophenothane
15. Gesarol
25. OMS 16
6. Dedelo
16. Guesapon
26. Pentachlorine
7. Dicophane
17. Gexarex
27. Rukseam
8. p,p’-DDT
18. Gyron
28. R50
9. Dichlorodiphenyltrichloroethane 19. Hildit
10. Dinocide
20. Ixodex
29. Zerdane
30. Zeidane
2.10 Formulations
Technical DDT has been formulated in many forms like solutions in
xylene or petroleum distillates, emulsifiable concentrates, water wettable
powders, granules, aerosols, smoke candles, charges for vaporisers and lotions
(Smith, 1991).
2.11 Toxicological effects
DDT is classified as "moderately hazardous" by WHO, based on the rat
oral LD50 of 113 mg/kg. Eating food with large amounts (grams) 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. The
same type of effects would be expected by breathing DDT particles in the air
or by contact of the skin with high amounts of DDT. Tests in laboratory
21
animals confirm the effect of DDT on the nervous system (ATSDR, 2002).
No effects have been reported in adults given small daily doses of DDT
by capsule for 18 months (up to 35 mg every day). People exposed for a long
time to small amounts of DDT (less than 20 mg per day), such as people who
worked in factories where DDT was made, had some minor changes in the
levels of liver enzymes in the blood. A study in humans showed that increasing
concentrations of p,p’-DDE in human breast milk were associated with
reductions in the duration of lactation. An additional study in humans found that
as the DDE levels in the blood of pregnant women increased, the chances of
having a pre-term baby also increased (ATSDR, 2002).
2.11.1 Toxicity to laboratory animals: Domenjoz (1944) described the
intoxification of DDT in animals. The animal becomes abnormally susceptible to
fear with violent reaction to normally sub- threshold stimuli. Motor unrest,
increased frequency of spontaneous movement, tremors and hyperirritability
were also observed.
Symptoms appear several hours after oral administration of the
compound, and death follows after 24-72 h. Cameron and Burgess (1945)
noticed sickness in rats, guinea pigs and rabbits.
The acute oral and dermal toxicity of DDT to common laboratory animals
is summarised in Table 2.7 (Smith, 1991).
Table 2.7: Acute oral and dermal L.D. 50 to animals.
Species
Rat
Mouse
Guinea pig
Formulation
Oral
Dermal
(mg/kg)
(mg/kg)
Water suspension or powder
500- 2500
1000
Oil solution
113- 450
250- 3000
Water suspension or powder
300- 1600
375
Oil solution
100- 800
250- 500
2000
1500
250- 560
1000
275
375
300- 1170
300- 2820
Water suspension or powder
Oil solution
Rabbit
Water suspension or powder
Oil solution
22
Dog
Oil solution
Cat
Water suspension or powder
> 300
100- 410
DDT is more toxic and readily absorbed orally when given as a solution
in vegetable oil or animal fat than when given in some petroleum fractions. It is
readily absorbed through the gastrointestinal tract, with increased absorption in
the presence of fats (ATSDR, Atlanta, 1994). One-time administration of DDT to
rats at doses of 50 mg/kg led to decreased thyroid function and a single dose of
150 mg/kg led to increased blood levels of liver-produced enzymes and
changes in the cellular chemistry in the central nervous system of monkeys
(Wasserman, 1982). Single doses of 50-160 mg/kg produced tremors in rats,
and single doses of 160 mg/kg produced hind leg paralysis in guinea pigs
(ATSDR, Atlanta, 1994). Convulsions were observed in mice following a oral
dose of two or more times the LD50 value. However, the onset of convulsions
was delayed by 6 h. when DDT was given orally at approximately the LD50 value
(Dale et. al., 1963). Single administrations of low doses to developing 10-day
old mice are reported to have caused subtle effects on their neurological
development (ASTDR, Atlanta, 1994). DDT is poorly absorbed by the dermal
route, with reported dermal LD50 of 2,500-3,000 mg/kg in female rats
(Wasserman, 1982; ASTDR, Atlanta, 1994), 1000 in guinea pigs (ATSDR,
Atlanta, 1994) and 300 in rabbits (ATSDR, Atlanta, 1994). It is not readily
absorbed through the skin unless it is in solution (ATSDR, Atlanta, 1994).
Inhalation of the DDT dust is not of special importance. Any of the
inhaled particle is deposited in the upper respiratory tract and is eventually
swallowed (Smith, 1991).
2.11.2 Biochemical effects: DDT acts on the nervous system membranes,
especially the axonal membranes. The effect on axons may be related to the
inhibition of Na+, K+ and Mg2+ - adenosine triphosphatase derived from a nerve
ending fraction of a rabbit brain that is inhibited by DDT (Smith, 1991). DDT is
known to cause prolonged opening of the ion gates of the sodium channel
23
perhaps by affecting phosphorylation in the α-subunit protein (Ishikawa et. al.,
1989).
DDT has also been proven to induce microsomal enzymes (Smith,
1991).
2.11.3 Respiratory effects: Rats fed a diet containing 20 mg DDT/kg/day for
27 months did not develop adverse respiratory effects with the exception of
squamous bronchial metaplasia in one rat (Deichmann et. al., 1967). In the 78week chronic bioassay conducted by NCI (1978), no adverse effects on the
respiratory system were observed in Osborne-Mendel rats treated in the diet
with up to 45 mg technical DDT/kg/day, 59 mg p,p’-DDE/kg/day, or 231 mg
technical DDD/kg/day. The same findings were reported for B6C3F1 mice
treated with up to 30.2 mg technical DDT/kg/day, 49 mg p,p’-DDE/kg/day, or
142 mg technical DDD/kg/day (NCI, 1978).
2.11.4 Endocrine effects: Exposure to DDT and DDT-related compounds,
particularly during development, can adversely affect the development and
function of the reproductive system of both female and male animals. This is
due primarily to the ability of some of these compounds to disrupt the action of
natural steroids and bind to receptors for estrogens and androgens.
2.11.5 Neurological effects: The nervous system appears to be one of the
primary target systems for DDT toxicity in humans after acute, high exposures.
A number of investigators conducted experimental studies on humans in the
1940s and 1950s at controlled doses that produced effects. Other data come
from accidental poisonings where dose levels were crudely estimated. Persons
exposed to 6 mg DDT/kg administered orally by capsule generally exhibited no
illness, but perspiration, headache, and nausea have been reported (Hayes,
1982). Convulsions in humans have been reported at doses of 16 mg DDT/kg
or higher (Hsieh, 1954). Velbinger (1947a, 1947b) exposed volunteers to oral
doses of 250, 500, 750, 1,000, or 1,500 mg DDT (approximately up to 22
mg/kg) suspended in an oil solution. Variable sensitivity of the mouth (defined
by the author as a prickle at the tip of the tongue, lower lip, and chin area) was
reported in volunteers exposed to 250 and 500 mg DDT/person. Six hours after
24
exposure to 750 or 1,000 mg DDT, disturbance of sensitivity of the lower part of
the face, uncertain gait, malaise, cold moist skin, and hypersensitivity to contact
were observed. Prickling of the tongue and around the mouth and nose,
disturbance of equilibrium, dizziness, confusion, tremors, malaise, headache,
fatigue, and severe vomiting were all observed in volunteers within 10 hours
after oral exposure to 1,500 mg DDT. All volunteers exposed to DDT orally had
achieved almost complete recovery from these acute effects within 24 hours
after exposure. Similar symptoms were reported in persons after accidental or
intentional ingestion of DDT (Francone, et. al., 1952; Garrett, 1947; Hsieh,
1954; Mulhens, 1946).
In humans, the nervous system appears to be one of the primary targets
in animals after acute, subchronic, and chronic oral exposure to DDT. Acute
oral exposure to high doses of DDT has been associated with DDT-induced
tremors or myoclonus (abrupt, repeated involuntary contractions of skeletal
muscles), hyperexcitability, and convulsions in several species. These effects
have been observed in rats after single oral gavage doses of 50–600 mg
DDT/kg/day (ATSDR, 2002). Mice receiving a single oral gavage dose of 160
mg DDT/kg had tremors (Hietanen and Vainio, 1976), and single doses of 200–
600 mg p,p’-DDT/kg/day induced convulsions (Matin et. al., 1981). In guinea
pigs and hamsters similarly dosed, no tremors were observed at 160 mg
DDT/kg, but hind leg paralysis occurred in guinea pigs (Hietanen and Vainio,
1976).
Acute oral exposure of animals to DDT has also been associated with
increases in brain biogenic amine and neurotransmitter levels. Alterations in the
metabolite 5-hydroxy-indoleacetic acid (5-HIAA), the degradation product of
serotonin, have been reported to correlate with DDT-induced tremors; doses at
50 mg/kg/day or greater resulted in increases in the levels of 5-HIAA in the
brain (ATSDR, 2002). Alterations in the levels of other neurotransmitters have
been found. The neurotransmitter changes observed are consistent with one of
the putative mechanisms for DDT toxicity; DDT is thought to influence
membrane ion fluxes and consequently potentiate neurotransmitter release.
25
Acetylcholine and norepinephrine decreased in rats after acute exposure to 400
mg/kg DDT (Hrdina et. al., 1973).
Also, aspartate and glutamine were
increased in brain tissue of rats (Hudson et. al., 1985; Hong et. al., 1986; Tilson
et. al., 1986).
2.11.6 Reproductive Effects: The potential association between DDT (and
DDT metabolites) and reproductive end points has been examined in numerous
studies (Leoni et. al., 1989; Gerhard et. al., 1998). Studies of women from India
reported that total DDT residues (DDT plus DDE and DDD) in maternal blood
from 25 full-term cases ranged from 7 to 73 ppb (mean 26 ppb) compared to
48–481 ppb (mean 66 ppb) in 15 pre-term and 90–1,280 ppb (mean 394 ppb) in
10 cases of spontaneous abortion (Saxena et. al., 1980, 1981). Mean DDT
residues in the respective placentas were 24, 66, and 162 ppb. Other
organochlorine pesticides were also elevated in the pre-term and abortion
cases relative to full-term cases.
Studies in humans suggest that high DDT/DDE burdens may be
associated with alterations in end points that are controlled by hormonal
function such as duration of lactation, maintenance of pregnancy, and fertility.
High blood levels of DDE during pregnancy have also been associated with
increased odds of having pre-term infants and small-for-gestational-age infants.
Perinatal exposure of animals to DDT/DDE has caused alterations in the
reproductive organs and infertility. Cohn and co-workers (2003) studied the
effects of DDT on the daughters of subjects previously exposed to DDT. Their
observations indicated that elevated levels of DDT in maternal blood were
clearly associated with decreased chance of pregnancy in their daughters.
2.11.7 Developmental Effects: The proper development of many systems and
functions depends on the timely action of hormones, particularly sex steroids;
therefore, interfering with such actions can lead to a wide array of effects that
may include altered metabolic, sexual, immune, and neurobehavioral functions.
In animals, DDT can produce embryotoxicity, fetotoxicity, and abnormal
development of the sex organs. Estrogen-like effects on the developing
reproductive system have been reported. The o,p’-isomers of DDT and DDE
26
have greater estrogen-like effects than other isomers and when administered to
rats in the first few days of life can seriously affect the development and
maturation of the reproductive system. Developmental effects have been
observed in animals after acute oral exposure to DDT during gestation or in the
early perinatal development period; the seriousness of these effects is
dependent on the isomeric form, the dose, and the timing of exposure.
Exposure during early gestation resulted in a 25% decrease in fetal body
weights and in significant decreases in fetal brain and kidney weights in the
offspring of pregnant rabbits given oral gavage doses of 1 mg DDT/kg/day on
gestation days 4–7 (Fabro et al., 1984). Offspring from rabbit dams orally
exposed to a dose level of $10 mg p,p’-DDT/kg/day by gavage on days 7–9 of
gestation showed a significant reduction in weight on day 28 (Hart et. al., 1971,
1972). However, treatment late in gestation (days 21, 22, and 23) did not induce
such an effect (Hart et. al., 1972). Gellert and Heinrichs (1975) exposed
pregnant rats orally to 28 mg o,p’-DDT, p,p’-DDT, o,p’-DDE, or o,p’-DDD/kg/day
on days 15–19 of gestation. No significant effects on body weight, weight of the
ovaries and pituitary, estrous cycle, or vaginal opening in the offspring were
noted with the exception of a small but significant delay (2 days) in vaginal
opening with the o,p’-DDD isomer. The p,p’-isomer is most prevalent in the
environment, accounting for approximately 85% of the total amount of DDT,
DDE or DDD found; also, technical-grade DDT contains between 65–80% p,p’DDT, between 15–21% o,p’-isomer, and up to 4% p,p’-DDD (Metcalf, 1995).
2.11.8 Cancer
The EPA classifies DDT, DDE, and DDD as a class B2 "probable" human
carcinogens. DDT is classified as a Group 2B, "possible" human carcinogen by
the International Agency for Research on Cancer (ATSDR, 2002).
2.11.8.1 Breast Cancer: Many epidemiological studies have investigated the
association between breast cancer and levels of DDT and DDT-derived
compounds in blood or adipose tissue from humans.
Some studies have
suggested a positive association (Falck et. al., 1992; Wolff et. al., 1993;
Dewailly et. al., 1994; Güttes et. al., 1998; Aronson et. al., 2000; Romieu et. al.,
27
2000), while others do not support such an association (Krieger et. al., 1994;
Lopez-Carrillo et. al., 1997; Schecter et. al., 1997; van’t Veer et. al., 1997;
Moysich et. al., 1998; Liljegren et. al., 1998; Dorgan et. al., 1999; Helzlsouer et.
al., 1999; Mendonca et. al., 1999; Zheng et. al., 1999, 2000; Ward et. al., 2000;
Wolff et. al., 2000a, 2000b; Demers et. al., 2000; Laden et. al., 2001). Much
higher levels of DDT have been reported in the blood of breast cancer patients,
as compared to healthy females (Mathur et. al., 2002). While individual studies
have come to conflicting conclusions, a recent review by Clapp et. al. (2008) of
all the evidence concludes that exposure to DDT before puberty increases the
risk of breast cancer later in life.
2.11.8.2 Pancreatic Cancer: Pancreatic cancer was weakly associated with
exposure to DDT in a nested case- control mortality study following up a cohort
of 5,886 chemical manufacturing workers who were potentially exposed
between 1948 and 1971 (Garabrant et al., 1992). The association between
pancreatic cancer and self-reported exposure to organochlorine pesticides
(DDT among them) was examined in group of 66 pancreas cancer cases and
131 controls in South Eastern Michigan (Fryzek et. al., 1997).
2.11.8.3 Prostate and Testicular Cancer: An ecologic study evaluated the
relationships between p,p’-DDE concentration in subcutaneous fat, or p,p’-DDE
in tree bark, and mortality from prostate and testicular cancers using
multivariate statistical techniques (Cocco and Benichou, 1998). Adipose DDE
was obtained from samples collected in the EPA Human Monitoring Program in
1968 for people in 22 states, tree bark DDE data were available for 18 states
representing the years 1992–1995, and age-adjusted mortality rates from
prostate and testicular cancers during 1971–1994 were available by state from
the National Center for Health Statistics. However, a recent study in the Journal
of the National Cancer Institute concluded that DDE exposure to may be
associated with testicular cancer. The incidence of seminoma in men with the
highest blood levels of DDE was almost double that of men with the lowest
levels of DDE (McGlynn et. al., 2008).
28
2.11.8.4 Other cancers: A Canadian study from 2007 found a positive
association between DDE and non-Hodgkin Lymphoma (Spinelli et. al., 2007).
A study of malaria workers who handled DDT occupationally found an elevated
risk of cancers of the liver and biliary tract. Another study has found a
correlation between DDE and liver cancer in white men, but not for women or
black men. An association between DDT exposure and pancreatic cancer has
been demonstrated in a few studies, but other studies have found no
association. Several studies have looked for associations between DDT and
multiple myeloma, and testicular, prostate, endometrial, and colorectal cancers,
but none conclusively demonstrated any association (Rogan and Chen, 2005).
In spite of some positive associations for some cancers within certain
subgroups of people, there is no clear evidence that exposure to DDT/DDE
causes cancer in humans.
2.11.9 Teratogenic Effects
DDT exposure is associated with early pregnancy loss, a type of
miscarriage. A prospective cohort study of Chinese textile workers by Altshul
(2005) found a positive, monotonic, exposure-response association between
preconception serum total DDT and the risk of subsequent early pregnancy
losses. Longnecker et. al. (2001) studied for the correlations between maternal
serum concentrations of DDT and DDE during pregnancy and certain hormone
regulated abnormalities (preterm labour, gonad formation, etc.) in their children.
They used blood collected almost 40 years ago from women who received
prenatal care from university hospitals. They demonstrated a steadily increasing
trend of preterm birth with serum DDE concentrations.
There is evidence that DDT causes teratogenic effects in test animals as
well. In mice, maternal doses of 26 mg/kg/day DDT from gestation through
lactation resulted in impaired learning performance in maze tests (ATSDR,
Atlanta, 1994). In a two-generational study of rats, 10 mg/kg/day resulted in
abnormal tail development (ATSDR, Atlanta, 2002). Epidemiological evidence
regarding the occurrence of teratogenic effects as a result of DDT exposure is
unavailable (ATSDR, Atlanta, 2002).
29
2.11.10 Mutagenic Effects
The evidence for mutagenicity and genotoxicity is contradictory.
Genotoxicity studies have been done in animals and in bacterial systems, but
studies in humans are limited. In pesticide sprayers who were exposed to DDT
as well as seven other pesticides, increased frequencies of sister chromatid
exchanges and chromosomal aberrations in peripheral lymphocytes were
reported, compared to controls (Rupa et. al., 1988).
Increases in sister
chromatid exchanges, the proliferation rate index, and the mitotic index were
also reported in pesticide sprayers exposed to DDT along with several other
pesticides (Rupa et. al., 1989).
Several workers have reported the genotoxic effects of DDT and
metabolites in animals. BALB/C mice exposed in vivo to DDT exhibited
chromosomal aberrations of the bone marrow (Johnson and Jalal, 1973; Larsen
and Jalal, 1974). Mahr and Miltenburger (1976) reported chromosomal damage
in the B14F28 Chinese hamster cell line after exposure to DDT, DDE, or DDD.
Palmer et. al. (1972) also observed these same results in kangaroo rat cells
(Potorus tridactylis) in vitro after exposure to DDT, DDE or DDD. Kelly-Garvert
and Legator (1973) reported a significant increase in chromosomal aberrations
in Chinese hamster V79 cells after exposure to DDE, but not DDT.
Collectively, the data do not suggest that DDT and related compounds
present a genotoxic hazard at environmentally relevant concentrations.
2.11.11 Fate in Humans & Animals
The intake of DDT and other pesticides in animals and human beings is
mainly through the biological transport. Once DDT enters the mammalian
systems,
it
is
very
slowly
transformed
into
1,1-dichloro-2,2-bis(p-
chlorophenyl)ethylene (DDE) and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane
(DDD), which are very readily stored in fatty tissues (ATSDR, Atlanta, 1994).
These compounds in turn are ultimately transformed into bis(dichlorodiphenyl)
acetic acid (DDA) via other metabolites at a very slow rate (ATSDR, Atlanta,
1994). DDA, or conjugates of DDA, are readily excreted via the urine (ATSDR,
Atlanta, 1994). In the early 1970s, the presence of DDT could be seen in the
30
human blood and fat tissue samples, which were collected from the developed
nations that banned DDT during 1970s. The level of DDT in the blood
decreased gradually towards the latter half of the 1970s (ATSDR, Atlanta,
1994), but the levels of DDT or metabolites may occur in fatty tissues at levels
of up to several hundred times that seen in the blood (ATSDR, Atlanta, 1994).
DDT or its metabolites may also be eliminated via mother’s milk by lactating
women (ATSDR, Atlanta, 2002). Several workers have reported the presence
of DDT in the breast milk (Rogan et. al., 1987; Bouwman, 1991; Bouwman et.
al., 1992; Gladen et. al., 1995).
2.12 Ecological effects
The 1972 EPA decision to ban DDT for most uses in the United States
was significantly influenced by a large body of scientific information
documenting adverse effects to wildlife (EPA, 1975). These observed effects
were severe, including the lethality of DDT to birds and fish and the DDEinduced reproductive effects in birds, particularly eggshell thinning (EPA, 1975).
Exposures for wildlife to DDT and its metabolites in the natural environment are
primarily associated with the accumulation and persistence of these
contaminants in both aquatic and terrestrial food chains. Ingestion of
contaminated food results in the deposition of DDT/DDE/DDD in tissues with
subsequent reproductive, developmental and neurological effects (ATSDR,
2002).
2.12.1 Effect on birds: DDT and its metabolites can lower the reproductive rate
of birds by causing eggshell thinning which leads to egg breakage, causing
embryo deaths. Sensitivity to DDT varies considerably according to species.
Predatory birds are the most sensitive. In the US, the bald eagle nearly became
extinct because of environmental exposure to DDT. According to research by
the World Wildlife Fund and the US EPA, birds in remote locations can be
affected by DDT contamination. Albatross in the Midway islands of the midPacific Ocean show classic signs of exposure to organochlorine chemicals,
including deformed embryos, eggshell thinning and a 3% reduction in nest
productivity. Researchers found levels of DDT in adults, chicks and eggs nearly
31
as high as levels found in bald eagles from the North American Great Lakes
(PANUPS, 1996). Laboratory studies on bird reproduction have demonstrated
the potential of DDT and DDE to cause subtle effects on courtship behaviour,
delays in pairing and egg laying and decreases in egg weight in ring doves and
Bengalese finches (WHO, environmental Health Criteria, 1989). DDT is also
reported to cause disturbance of endocrine system, resulting in thyroid
dysfunction,
decreases
fertility,
metabolic
abnormalities,
behavioural
abnormalities etc. (Colborn and Thayer, 2000).
2.12.2 Effect on fish: DDT is highly toxic to fish. The 96-hour LC50 ranges from
1.5 µg/litre for the largemouth bass to 56 µg/litre for guppy. The reported 96hour LC50s in fathead minnow and channel catfish are 21.5 µg/L and 12.2 µg/L
respectively (Johnson and Finlet, 1980). Smaller fish are more susceptible than
larger ones of the same species. An increase in temperature decreases the
toxicity of DDT to fish (PANUPS, 1996). Koeman (1979) has reported cases of
vertebral fractures and symptoms of vertebral and spinal deformation of fish.
Attri (1981) has reported that the sublethal concentrations can cause
reproductive abnormalities. In addition to acute toxic effects, DDT may
bioaccumulate significantly in fish and other aquatic species, leading to longterm exposure. This occurs mainly through uptake from sediment and water into
aquatic flora and fauna, and also fish (WHO, environmental Health Criteria,
1989). Fish uptake of DDT from the water will be size-dependent with smaller
fish taking up relatively more than larger fish (WHO, environmental Health
Criteria, 1989).
DDT also causes demasculinisation and feminisation of male fish,
defeminisation and masculinisation of female fish, gross birth deformities in fish
and turtle (Colborn and Thayer, 2000).
2.12.2 Effect on plants: The effect of DDT on plants has been studied by
Deepthi et. al. (2005). They have reported the toxic effect of DDT on the
germination of some seeds. There was a marked reduction in the germination of
Brassica juncea,
Raphanus sativus, Phaseolus aureus, Phaseolus vulgaris,
Hibiscus esculentum, Oryza sativa and Eleucine coracona with increase in DDT
32
concentration.
Abnormalities
were
observed
in
root,
shoot
and
leaf
developments. Enzyme activities were also affected.
2.13 Regulatory status
Rachel Carson’s Silent Spring in 1962 sounded the initial alarm against
DDT. The environmentalists also blamed DDT. Finally in 1972, the U. S.
Environmental Protection Agency issued a cancellation order for DDT based on
adverse environmental effects of its use, such as those to wildlife, as well as
DDT’s potential human health risks (USEPA, 2008). DDT was first banned in
Hungary in 1968 then in Norway and Sweden in 1970 and the US in 1972, but
was not banned in the United Kingdom until 1984. It was banned in India for
agriculture in April 1997 but is still permitted to be used for health programmes.
But because of the cost and ineffectiveness of other insecticides in controlling
malaria, no other insecticide proved as an alternative to DDT. Attaran (2001)
pointed the fact that malaria rebounded in Africa and Asia when DDT phasing
out started. Thus, finally, 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 (Dash et. al., 2007).
2.14 Detection of DDT
The use of pesticides has become an indispensable tool in agriculture for
the control of pests and in public health programmes for the eradication of
vector borne diseases. Less than 1% of the applied pesticide generally reached
the target pests (Pimentel, 1983). Therefore most of the pesticide remained
unused and entered into the ecosystem. These excessive pesticide residues
accumulate in the biosphere and create ecological stress. Soil and water are
the ultimate sinks for the excessive pesticides. In India, pesticide residues can
still be detected in a wide range of food stuffs including cereals, oilseeds,
spices, vegetables, butter and milk (Skeritt et. al., 2003). Hence, the monitoring
33
and detection of these environmental contaminants are of prime importance. A
number of detection techniques are available. These may be classified broadly
into two categories:
A. Chromatographic techniques
B. Non-chromatographic techniques
2.14.1 Chromatographic techniques: The chromatographic techniques
include the thin layer chromatography (TLC), high performance thin layer
chromatography (HPTLC), gas chromatography (GC) and high performance
liquid chromatography (HPLC) which find wide applications in qualitative and
quantitative analyses of pesticides in foods, environmental samples such as soil
and drinking water and in biological samples (Zambonin et. al., 2004; Sherma,
2005; Tagami et. al., 2007). Pesticide determinations are performed on silica
gel TLC or HPTLC commercially pre-coated plates. TLC and HPTLC provided
high sample throughput and simplicity of assay procedure. The assay could be
completed by developing the spotted plates in a TLC chamber containing a
suitable mobile phase. After the mobile phase reached 3/4th of the plate, a
suitable chromogen can be sprayed to the air- dried plates. Pesticide
identification is based on comparing RF values between sample and standard
zones. Quantitative TLC of pesticides is performed by measuring the visible
absorption, UV absorption, or fluorescence of standard and sample analyte
zones in- situ on a high performance layer using a slit- scanning densitometer in
the reflection mode (Sherma, 2005).
Karanth et. al. (1999) have detected isomers of HCH using TLC. They
have detected DDT, aldrin, chlordane, endosulfan and HCH- isomers by a
simple, one- step chromatographic method called finger printing technique in
market samples of fresh vegetables obtained from Mysore city. But both the
methods could detect only ppm levels of the compound and required sample
cleanup.
Tilak et. al. (2003) detected pyrethroid insecticide fenvalerate by
impregnating silver nitrate reagent into silica gel layer prior to development of
the plate with hexane- acetone (3:1) during toxicity and residue studies with the
34
freshwater fish Channa punctatus. This was followed by exposure to UV light for
about 10 min to allow observation of dark zones on a light brown background.
Shrivastava and Vaishnav (2003) reported the use of combination of
aminoacid- pesticide combination for the detection of commonly used pesticides
using soil- TLC. Mobility was found to increase or decrease uniquely for each
aminoacid- pesticide combination.
Gas chromatography is another chromatographic technique that is widely
used in pesticide analyses. Brewerton (1969) used the technique to reveal the
accumulation of DDT in the fat of animals. An
63
Ni selective electron capture
detector was used in the detection of organochlorine and organophosphorus
pesticides in blood samples collected from 4 different villages of Punjab (Centre
for Science and Environment, 2005). Deerasamee and Tiensing (2007) have
reported the detection of organophosphorus pesticide degradation in water by
using a flame photometric detector. Tagami et. al. (2007) have reported the
analysis of 17 organochlorine pesticides in natural medicines by GC/ MS with
negative chemical ionisation. The assessment of organophosphorus pesticide
residues in wine and fruit juices has been done using solid phase
microextraction- GCMS (Zambonin et. al., 2004). Mercer (2005) used gas
chromatography with a mass selective detector (GC–MSD), electron impact
ionization, and selected-ion monitoring to detect halogenated pesticides. The
method could detect upto 50 ng/g levels of the pesticides in a variety of fruit and
vegetable matrixes. The GC method could detect upto 10ppb levels of the
samples. But the method required extensive sample cleanup and trained
personnel to handle the equipment.
2.14.2 Non- chromatographic techniques: Non- chromatographic methods for
residue detection consist of a wide variety of techniques. These may be divided
into ‘physical’- and ‘biological’- based methods, based on whether or not
biological reagents are involved. Among the physical techniques that fit this
category are spectrophotometry, radioactivity, fluorescence, luminescence,
phosphorescence and voltametry. Biological techniques include immunoassays,
biosensors, bioassays, enzyme assays and polymerase chain reaction (PCR).
35
2.14.2.1 Immunoassays for pesticides: Immunoassay for pesticides was first
reported by Centeno et. al. (1970) when they developed antibodies that
selectively bound malathion. A few years later, radioimmunoassays were
developed for aldrin and dieldrin and for parathion. In 1972, Engvall and
Perlmann (1972) introduced the use of enzymes as labels for immunoassay and
launched the term enzyme-linked immunosorbent assay (ELISA). In 1980,
Hammock and Mumma (1980) described the potential for ELISA for
agrochemicals and environmental pollutants. Since then, the use of
immunoassay for pesticide analysis has increased dramatically. Table 2.8
summarises the immunoassays developed for several pesticides by different
co- workers.
Table 2.8: Immunoassays for pesticides.
Pesticide
Test format
Reference
Acetochlor
Enzyme immunoassay
Hennion, 1998
Feng et. al., 1992; Lawruk et.
Alachlor
Enzyme immunoassay
al., 1992; Sharp et. al., 1992;
Hennion, 1998;
Gabaldon et. al., 1999.
Amitrole
Enzyme immunoassay
Jung et. al., 1991.
Hennion, 1998; Gabaldon et.
Aldicarb
Enzyme immunoassay
al., 1999; Brady et. al., 1989;
Itak et al., 1992.
Aldrin
Atrazine
Radioimmunoassay
Langone and Van Vunakis,
1975.
Enzyme immunoassay
Dankwardt, 2001.
Piezoelectric immunoassay
Steegborn and Skladal, 1997.
Dipstick immunoassay
Mosiello et. al., 1998,
Wittmann et. al., 1996.
Carbaryl
Enzyme immunoassay
Abad and Montoya, 1994;
36
Marco et. al., 1993.
Carbofuran
Enzyme immunoassay
Jourdan et al., 1995;
Razak, et. al., 1998.
Manclus and Montoya, 1995;
Chlorpyriphos
Enzyme immunoassay
Gabaldon et. al., 1999;
Hennion, 1998.
Gabaldon et. al., 1999;
Cyclodiene
Enzyme immunoassay
DDA
Enzyme immunoassay
Banerjee, 1987.
Enzyme immunoassay
Abad et al., 1997;
Manclus et. al., 2004.
Hirano et. al., 2008;
Maestroni et. al., 2001;
Amitarani et. al., 2002;
Amitarani et. al., 2001;
Karanth et. al., 1999;
Anfossi et. al., 2004.
DDT
Indirect competitive ELISA
Hochel and Musil, 2002.
Electrochemical ELISA
Valentini et. al., 2003.
Optical immunosensor
Mauriz et. al., 2007.
assay
Chemiluminiscent flow IA
Botchkareva et. al., 2002.
Nanomechanical biosensor
Alvarez et. al., 2003.
DDE
Enzyme immunoassay
Shivaramaiah et. al., 2002.
Diazinon
Enzyme immunoassay
Hennion, 1998.
Dieldrin
Radioimmunoassay
Langone and
Van Vunakis, 1975.
Dreher and Podratzki, 1988;
Endosulfan
Enzyme immunoassay
Lee et. al., 1995;
Reck and Frevert, 1990.
Heptachlor
Enzyme immunoassay
Stanker et. al., 1990.
37
Enzyme immunoassay
Hennion, 1998;
Kolosova et. al., 2004.
Methyl
parathion
Fluorescence polarisation
Kolosova et. al., 2004.
IA
Immunochemiluminiscence
Chouhan et. al., 2006.
assay
Flow immunosorbent assay
Kumar et. al., 2006.
Enzyme immunoassay
Ferguson and Larkin, 1994;
Wong et. al., 1991.
Parathion
Paraxon
Radioimmunoassay
Ercegovich et. al., 1981.
Enzyme immunoassay
Brimfield et. al., 1985;
Heldman et. al., 1985;
Hunter and Lenz, 1982.
Radioimmunoassay
PCP
Enzyme immunoassay
Heldman et. al.,1985.
Hall et. al.,1992;
Mapes et. al.,1992.
Bonwick et. al.,1994;
Permethrin
Enzyme immunoassay
Stanker et. al.,1989;
Skerritt et. al.,1992.
Triazine
Enzyme immunoassay
Gabaldon et. al., 1999;
Hennion, 1998.
Immunoassay provides a simple, powerful, fast and inexpensive
detection method with enormous potential which includes the generation of
quantitative data. The method can be used for monitoring of large number of
analytes, including low- molecular- mass- pesticide molecules. The technique is
now gaining acceptance and competing with other analytical methods. The
technique is useful in the analysis of ground and drinking water, where matrix
effects are rarely encountered. But with other food and environmental samples,
matrix effect needs to be removed by a suitable clean- up method. Many
detection kits have been developed for the detection of various pesticides. But
38
the long- term storage under environmental conditions and their repeated use
with complex sample matrices is a challenge. Finally, new strategies to increase
sensitivity of the assay need to be developed.
2.15 Degradation of DDT
Xenobiotic compounds are anthropogenic chemicals that are present in
the environment at unnaturally high concentrations. The xenobiotic compounds
are either not produced naturally, or are produced at much lower
concentrations. These compounds are recalcitrant and persistent. The
recalcitrance of these compounds may be due to one or more of the following
reasons:
i.
They are not recognised as substrate by the existing degradative enzymes.
ii.
They are highly stable, i.e., chemically and biologically inert due to the
presence of
substitution groups like halogens, nitro-, sulphonate, amino-,
methoxy- and carbamyl groups.
iii.
They are sparingly soluble in water, or are adsorbed to external matrices like
soil.
iv.
They are highly toxic or give rise to toxic products due to microbial activity.
v.
Their large molecular size prevents entry into microbial cells.
vi.
Inability of the compounds to induce the synthesis of degrading enzymes.
vii.
Lack of the permease needed for their transport into the microbial cells.
Among the xenobiotics, polyaromatic, chlorinated and nitroaromatic
compounds were shown to be toxic, mutagenic and carcinogenic as discussed
under section 2.10. DDT and related compounds have been classified under
POPs by the Stockholm convention in 2001. These compounds are recalcitrant
and are persistent in the environment for many years. Persistence of some of
these compounds is given in Table 2.9.
39
Table 2.9: Persistence of some organochlorines in soil.
Compound
Approximate duration of persistence (years)
PCBs
>30
Chlordane
>12
HCH
>10
DDT
>10
Aldrin
>9
Heptachlor
>9
Toxaphene
>6
Dicamba
2
(Source: Manonmani and Kunhi, 1999)
The uptake of these compounds can lead to their accumulation in the
food chain. The removal of these compounds from the environment is therefore
a priority. The compounds may be subjected to enzymatic or non- enzymatic
reactions brought about by the inhabitants of the environment. Remediation
includes photochemical remediation, phytoremediation, chemical remediation
and microbial remediation.
Chemical remediation: Chemical reactions bring about changes in the
structure of the compound. Presence of metals and metal ions such as iron,
aluminium, etc. make the pesticides susceptible to various chemical reactions.
The alkalinity of the medium also plays an important role in destabilising many
pesticides. But chemical decomposition of pesticides is slow and requires a
large investment to improve the process speed.
Photochemical remediation: The photolysis of the chemicals may be
significant only on the surface soil or on the plant surface other than in
atmosphere (Lakshmi et. al., 2002). The photochemical reactions may be
significant in aquatic environments than in soil (Alexander, 1981). This is due to
the fact that the penetration of sunlight is more in aquatic environment than in
soil. Furthermore, most pesticides are applied to established crops where the
foliar cover can drastically reduce the intensity of light reaching the soil (Hill and
Wright, 1978). It rarely occurs under natural conditions and would be very slow.
40
Bioremediation: Bioremediation involves the use of microbes or plants
to detoxify and degrade environmental contaminants.
Phytoremediation: Phytoremediation involves the use of vascular plants
and algae to remove and control waste or spur waste breakdown by
microorganisms in the soil zone that surrounds and is influenced by the roots of
plants. The diverse wastes that can be managed by using phytoremediation
include xenobiotic organic chemicals, sewage, salts, nutrients, heavy metals,
metalloids, and air pollutants. Phytoremediation may be applied wherever the
soil or static water environment has become polluted. Several workers have
reported the use of plants for the remediation of contaminants. Schröder et. al.
(2008) have reported the use of Phragmites australis plants to accumulate
organic xenobiotics in their rhizomes. Kawahigashi et al. (2008) reported the
use of rice plants for the remediation of atrazine and metolachlor from soil.
Mercury, selenium and organic pollutants such as polychlorinated biphenyls
(PCBs) have been removed from soils by transgenic plants containing genes for
bacterial enzymes (Meagher, 2000). Eapen et. al. (2007) have reported the use
of transgenic plants with enhanced potential for detoxification of xenobiotics
such as trichloro ethylene, pentachlorophenol, trinitro toluene, glycerol trinitrate,
atrazine, ethylene dibromide, metolachlor and hexahydro-1,3,5-trinitro-1,3,5triazine. Phytodegradation of DDT by aquatic plant Elodea Canadensis and a
terrestrial plant Pueraria thunbergiana was reported by Garrison et. al. (2000).
Phytoremediation
is
a
clean,
efficient,
inexpensive
and
non-
environmentally disruptive method, as opposed to processes that require
excavation of soil. It is a cost- effective and safe method in which the plants can
be easily monitored. Limitations of phytoremediation include the presence of
hazardous metabolites in the food chain. The slow growth, low biomass and
leaching of contaminants into groundwater are also responsible for the limited
use of phytoremediation. Phytoremediation is limited to the surface area and
depth occupied by the roots.
41
Bioremediation: Mineralisation of an organic molecule in water and soil is
almost always a consequence of microbial activity. During the conversion of the
organic substrate to inorganic products, the microbes make use of some carbon
in the substrate and convert it to cell constituents. Energy is released and
population of the microbes increase. Thus, mineralisation is a growth- linked
process.
Bioremediation is an effective biotechnological approach to cleanup a
polluted environment. The approaches to bioremediation are environmental
modification, such as through nutrient application, aeration and addition of
appropriate degraders by seeding (Iwamoto and Nasu, 2001). Bioremediation is
primarily based on either microorganisms naturally present at the sites, or on
microbial inoculants developed in the laboratory and introduced at the site.
Microbial remediation of soils and ground water can be done either in- situ or
ex- situ.
In-situ bioremediation treats the contaminated soil or groundwater in the
location in which it is found. In this technology, oxygen and occasionally
nutrients are pumped under pressure into the soil through wells. The nutrients
are spread on the surface to infiltrate into the contaminated area of material or
the saturated zone. Iwamoto and Nasu (2001) have classified in-situ
bioremediation processes into three categories:
Bioattenuation- A method of monitoring the natural progress of
degradation to ensure that contaminant concentration decreases with time at
relevant sampling points.
Biostimulation- A method of manipulating the environment to stimulate
the bioremediation and increase the reaction rate where natural degradation is
slow or does not occur. Biostimulation can be done by supplying the
environment with nutrients.
Bioaugmentation- A method of enhancing the biodegradative capacities
of contaminated sites by inoculation of bacteria with desired catalytic
capabilities. Since large amounts of bacteria are added to contaminated sites,
the effect of bacteria on both human and environment must be clarified in
42
advance. The effect of inoculated bacteria on the indigenous population also
needs to be confirmed. Nakamura et. al. (2000) conducted the first field
experiment in Japan wherein a phenol utilising bacteria, Ralstonia eutropha KT1, isolated from the same contaminated site was injected without adding any
substrates.
In-situ bioremediation offers several advantages over physical and
chemical remediation. It is cost- effective, can be done on- site with minimum
site disruption, eliminates transport cost and eliminates waste permanently.
Ex-situ bioremediation are the treatments that remove contaminants at a
separate treatment facility. This requires pumping of the groundwater or
excavation of contaminated soil prior to remediation treatments. After
remediation, the remediated soil is brought back to the site and refilled.
Carberry et. al. (1991) described a controlled land farming technique to bioremediate petroleum contaminated soil. The contaminated soil was placed in a
green house on a plastic sheet to a depth of 18 inches. The soil was periodically
stirred for aeration and nutrients and microorganisms were also added
periodically. pH could also be controlled in such a case with lot of ease. Water
can also be added to maintain the moisture content of the soil.
Ex-situ bioremediation can be further broken down into two main
components or processes; slurry-phase and solid-phase treatment.
Slurry-phase-
This treatment involves the initial combination of water
with the contaminated soil and later degradation in a bioreactor. Biodegradation
in a slurry- phase reactor has been shown to be effective in treating highly
contaminated soils and sludge with contaminant concentrations of 2,5002,50,000ppm (Ritter and Scarborough, 1995).
Solid-phase-
This treatment achieves the similar goal of the former
treatment yet, in this process, the contaminated soil is placed in a bed and
nourished with nutrients, moisture and oxygen in hopes that decomposition will
occur.
Although many strategies are available to get rid of these toxic
contaminants from the environment, microbial remediation seems to be one of
43
the most environment friendly, safe and cost- effective methods. A wide variety
of pesticides can be degraded by microbes even under mild conditions
compared with chemical or physical degradation. Many reports are available on
the
microbial
degradation
(microbial
bioremediation)
of
environmental
pollutants.
2.15.1 Microbial remediation
Even with the awareness about the ill effects of pesticides, the use of
pesticides has been indispensable. The use of pesticides in public health and
agriculture has led to its bioaccumulation. Thus, removal of pesticides from the
environment is important. Microbes play an essential role in the bioconversion
and total breakdown of pesticides. Among the microbial communities, bacteria
and fungi are the major degraders of pesticides. Yeasts, microalgae and
protozoa are less frequently encountered in the degradation process. Microbes
responsible for the degradation of various pesticides have been described in
Table 2.10. Among bacteria, Pseudomonads are considered to be the most
efficient group in bioremediation. Table 2.11 describes the bioconversion of
xenobiotics affected by Pseudomonads.
Table 2.10: Microorganisms responsible for pesticide degradation.
Pesticide
Microorganism
Reference
Chlorophenoxy acids
Alcaligenes eutrophus
Alcaligenes xylosoxidans
2,4-D
Flavobacterium sp. 50001
(1981)
Gunulan and Fournieer
(1993)
Chaudhry and
Huang(1988)
Pseudomonas putida
Lillis et al (1983)
Pseudomonas cepacia
Kilbane et al (1982)
Comamonas sp.
2,4,5-T
Don and Pemberton
Pseudomonas cepacia
Bulinski and Nakatsu
(1998)
Karns et. al. (1982)
44
DPA
Mecoprop
Mecocarp
Flavobacterium sp.
Sphingomonas
herbicidivorans MH
Alcaligenes denitrificans
Horvath et. al. (1990)
Zipper et al (1966)
Tett et. al. (1997)
Organochlorines
Aerobacter aerogenes
Wedemeyer (1966)
Alcaligenes eutrophus A5
Nadeau et. al. (1994)
Agrobacterium
tumefaciens
DDT
Johnson et al (1967)
Arthrobacter sp.
Patil et. al. (1967)
Bacillus cereus
Johnson et. al. (1967)
Bacillus cooagulans
Langlois et. al. (1970)
Bacillus megaterium
Plimmer et. al. (1968)
Bacillus subtilis
Johnson et. al. (1967)
Clostridium pasteurianum
Johnson et. al. (1967)
Clostridium michiganense
Johnson et al (1967)
Enterobacter aerogenes
Langlois et. al. (1970)
Erwinia amylovora
Johnson et. al. (1967)
Escherichia coli
Langlois et. al. (1970)
Hydrogenomonas sp.
Focht and Alexander
(1970)
Klebsiella pneumoniae
Wedemeyer (1966)
Kurthia zapfii
Johnson et. al. (1967)
Micrococcus sp.
Plimmer et. al. (1968)
Nocardiai sp.
Chacko et. al. (1996)
Pseudomonas
Bidlan and Manonmani
aeruginosa DT-Ct1
(2002)
Pseudomonas
Bidlan and Manonmani
aeruginosa DT-Ct2
(2002)
45
DDT
Pseudomonas
Bidlan and Manonmani
fluorescens DT-2
(2002)
Serratia marcescens
Mendel and Walton (1966)
Serratia marcescens DT-
Bidlan and Manonmani
1P
(2002)
Streptomyces
annomoneus
Streptomyces
aureofacians
Streptomyces
viridochromogens
Xanthomonas sp.
Phanerochaete
chrysosporium (fungus)
Chacko et. al. (1996)
Chacko et. al. (1996)
Johnson et. al. (1967)
Bumpus and Aust (1987)
Trichoderma viride
Matsumura and Boush
(fungus)
(1968)
Aerobacter aerogenes
Bacillus cereus
Bacillus megaaterium
γ-HCH
Chacko et. al. (1996)
Mecksongsee and Guthrie
(1965)
Mecksongsee and Guthrie
(1965)
Mecksongsee and Guthrie
(1965)
Citrobacter freundii
Jagnow et. al. (1977)
Clostridium rectum
Jagnow et. al. (1977)
Escherichia coli
Francis et. al. (1975)
Pseudomonas
Mecksongsee and Guthrie
fluorescens
(1965)
Pseudomonas putida
Benzet and Matsumara
(1973)
46
Pseudomonas
paucimobilis
γ-HCH
Pseudomonas sp.
Anabaena sp.
(Cyanobacteria)
Nostocellipssosun
(Cyanobacterium)
Phaenrochaete
chrysosporium (fungus)
Trametesversicolor
(fungus)
Phanerochaete sordida
(fungus)
Cyathus bulleri (Fungus)
Bachmann et. al. (1988)
Sahu et. al. (1990)
Kurtiz and Wolk (1995)
Kurtiz and Wolk (1995)
Mougin et. al. (1996)
Singh and Kuhad (1999a)
Singh and Kuhad (1999b)
Singh and Kuhad (1999b)
Organophosphates
Flavobacterium sp
Pseudomonas
aeruginosa
Parathion
(1973)
Gibson and Brown (1974)
Pseudomonas diminuta
Serdar et. al. (1982)
Pseudomonas
Boush and Matsumura
melophthara
(1967)
Pseudomonas stutzeri
Phorate
Sethunathan and Yoshida
Doughton and Hsieh
(1967)
Rhizobium japonium
Mich and Dahm (1970)
Rhizobium melioloti
Mich and Dahm (1970)
Streptomyces lividans
Steiert et. al. (1989)
Bacillus megaterium
La Partourel and Wright
(1976)
47
Carbamates
Pseudomonas cepacia
Bousch and Matsumura
melophthora
(1967)
Pseudomonas
Chapalamadugu and
aeruginosa
Chaudhry (1993)
(Fungi)
Aspergillus flavus (Fungi)
Aspergillus terreus
(Fungi)
Carbofuran
Liu and Bollog (1971)
Bollog and Liu (1972)
Bollog and Liu (1972)
Culcitalna sp. (Fungi)
Sikka et. al. (1975)
Halosphaeria sp. (Fungi)
Sikka et. al. (1975)
Fusarium solani (Fungi)
Bollog and Liu (1972)
Rhizopus sp. (Fungi)
Bollog and Liu (1972)
Penicillium sp. (Fungi)
Bollog and Liu (1972)
Achromobacter sp. WMIII
Karns et. al. (1986)
Arthrobacter sp.
Ramanand et. al. (1988)
Flavobacterium sp.
Chaudhry and Ali (1988)
Pseudomonas cepacia
Pseudomonas stutzeri
Bacillus pumilis
s-Triazines
(1980)
Pseudomonas
Gliocladium roseum
Carbaryl
Venkateswarlu et. al.
Venkateswarlu et. al.
(1980)
Mohapatra and Awasthi
(1977)
Mohapatra and Awasthi
(1977)
Pseudomonas sp.
Cook and Hutter (1981)
Klebsiella pheumoniae
Cook and Hutter (1981)
Rhodococcus corallinus
Cook and Hutter (1981)
Rhizobium sp. PATR
Bauguard et. al. I(1997)
48
Phanerochaete
chrysosporium (Fungus)
Mougine et. al. (1994)
Source: Singh et. al., 1999.
Table 2.11: Bioconversion of xenobiotics effected by Pseudomonads.
Mode of action
Species
Hydrolysis of carbaryl, dichlorphos, diazinon, parathion
Ps.melophthora
Hydrolysis of parathion
Ps.stutzeri
Dehalogenation of halide acetate
Ps. species
Total dehalogenation of DDT, aromatic ring cleavage
Ps.aeruginosa
Total degradation of 3- chlorobenzoate
Ps.putida
Oxidative dehalogenation of lindane
Ps.putida
Reduction of nitro group in 4,6- dinitro-q- cresol
Ps. species
Total degradation of 2,4,5- T
Ps.sepacia
Degradation of toluene, xylene, styrene, -
Ps.putida
methylstyrene
Ps.aeruginosa
Source: Golovleva et. al., 1990.
The available literature on microbial degradation of xenobiotics indicates
that many studies have mainly considered two aspects: (1) the fundamental
basis of biodegradation activities, the evolution and transformation of such
activities among microbes. (2) Bioremediation techniques to detoxify severely
pesticide-contaminated environments (Kumar et. al., 1996). For bioremediation
technologies to work, all the physiological, microbiological and biochemical
aspects involved in the pollutant transformation, have to be considered (Singh
et. al., 1999).
Several microorganisms are known to degrade DDT anaerobically
(Wedemeyer, 1966). The primary metabolic mechanism that was studied was
the reductive dechlorination of DDT, with the formation of DDD (1,1-dichloro2,2-bis(4-chlorophenyl)ethane or dichlorodiphenyldiichloroethane) (Kallman and
Andrews, 1963; Barker and Morrison, 1964). This degradation was later
determined to be microbial and Proteus vulgaris was isolated (Barker et. al.,
1965) which could degrade DDT mainly to DDD. Nadeau et. al. (1994) has
49
reported the aerobic degradation of DDT via 4- chlorobenzoic acid by
Alkaligenes eutrophus A5. Cell free extracts of Escherichia coli, Klebsiella
pneumoniae and Enterobacter aerogenes are dechlorinated p,p -DDT to DDE
anaerobically (Singh et. al., 1999). Bumpus and Aust (1987) reported the
degradation of DDT by the white rot fungus, Phanerochaete chrysosporium.
Chacko et. al. (1966) isolated numerous actinomycetes (Nocardia sp.,
Streptomyces
aureofaciens,
Streptomyces
cinnamoneus,
Streptomyces
viridochromogenes) from soil, which readily degraded DDT to DDD. These
organisms however, required another carbon source to facilitate degradation.
Soil fungi not only produced DDD and small amounts of dicofol (4,4’-dichloro-a(trichloromethyl) benzhydrol), but some variants could produce DDA (bis(4chlorophenyl)acetic acid) or DDE (1,1-dichloro-2,2-bis(4-chlorophenyl)ethene)
exclusively (Matsumura and Boush, 1968). Wedemeyer (1967) reported
dehalogenation of DDT to various metabolites under anaerobic conditions by
Aerobacter aerogenes. DDD was obtained under both aerobic as well as
anaerobic conditions when DDT was incubated with Aerobacter aerogenes
(Mendel et. al., 1967; Wedemeyer, 1967). Escherichia coli dechlorinated 50% of
DDT to DDE when grown in various broths or skimmed milk (Langlois, 1967).
Under aerobic conditions the major product of DDT metabolism, in Bacillus
cereus, B. coagulans, B. subtilis, was DDD while DDMU (1-chloro-2,2-bis(4chlorophenyl)ethylene), DDMS (1-chloro-2,2-bis(4-chlorophenyl)ethane), DDNU
(2,2-bis4-chlorophenyl)ethane), DDOH (2,2-bis(4-chlorophenyl)ethanol), DDA
and DBP (4,4’-dichlorobenzo phenone) were in trace amounts and were found
under anaerobic conditions (Langlois et. al., 1970). Hydrogenomonas sp.
yielded DDD, DDMS, DDMU, DBH (4,4’-dichlorobenzhydrol), DDM (bis(4chlorophenyl)methane) and DDA (Focht and Alexander, 1970). DDD was
further
degraded
through
dechlorination,
dehydrochlorination
and
decarboxylation to DBP or to a more reduced form, DDM.
Many workers have reported the addition of external inoculum to soil for
the degradation of DDT, which is described in Table 2.12.
50
Table 2.12: Soil studies on DDT degradation with external inoculum added
to the soil.
Organism
Reference
Remarks
Enterobacter aerogenes Kearney et. al., 1969
Phanerochaete
Barr and Aust, 1994
DDT to DDE
Fernando et. al., 1989
Added to soil with corn
chrysosporium
Phanerochaete
chrysosporium
cob mixture, degraded
14
C- DDT in 60 days by
39 %
Phanerochaete
Katayama and
Added to the soil with
chrysosporium
Matsumura,1991.
UV exposure
Alcaligenes eutrophus
Nadeau et. al., 1994.
Pseudomonas
Manonmani et. al., 2000 IP # 226/DEL/2000
aeruginosa
Serratia marcescens
Bidlan and Manonmani,
Aerobic degradation of
DT-1P
2002.
DDT.
Serratia marcescens
Bidlan, 2003.
Degradation of DDT in
DT-1P
Pseudomonas
soil.
Bidlan, 2003.
aeruginosa DT- Ct- 1
Pseudomonas
soil.
Bidlan, 2003.
aeruginosa DT- Ct- 2
Pseudomonas
fluorescens DT- 2
Degradation of DDT in
Degradation of DDT in
soil.
Bidlan, 2003.
Degradation of DDT in
soil.
Various factors affecting DDT degradation has been described in Table
2.13.
51
Table 2.13: Factors affecting DDT degradation
Reference
Remarks
Guenzi and Beard, 1968
Burge, 1971
Mitra and Raghu, 1988
Guenzi and Beard, 1967
Castro and Yoshida, 1971
Farmer et. al., 1974
Readily available carbon sources
enhanced DDT conversion to DDD
under anaerobic conditions
Green manuring tends to decrease the
persistency of DDT
Flooding resulted in faster degradation
of DDT
Boul, 1996
Xu et. al., 1994
Flooding showed slower degradation
Nair et. al., 1992
Slower degradation in clay surface and
Boul, 1995
organic matter
Reported treatment of DDT-
Keller and Rickabaugh, 1992
Parfitt et. al., 1995
contaminated soil. Soil with surfactants
released DDT from its surface,
enhancing aqueous solubility
Kumar et. al., 1996
Anaerobic degradation of - HCh
occurred rapidly in paddy soil.
Source: Bidlan, 2003.
Microorganisms play a vital role in the environmental fate of pesticides,
leading to the complete detoxification and mineralisation of the contaminant.
Microorganisms
are
associated
with
enhanced
degradation.
Microbial
degradation is cost- effective and hence degradation using microbes is gaining
a lot of importance now. The major concern is the bacterial interactions with the
native microflora and the environmental factors. This can also be taken care by
selecting the microbe from the same contaminated site that has to be
remediated.
52
2.16 Cloning of xenobiotic degrading genes
The wide dispersion of toxic chemicals throughout the varied soil and
water structures of our earth has stimulated the development of bioremediation
technologies which may be applied to different environmental situations. One of
the new developing technologies involves the genetic engineering of
microorganisms with enhanced degradative capabilities for bioaugmentation of
selected, contaminated environments.
Many xenobiotics contain functional groups and partial structures not
usually found in nature. Many pesticides are bulky complex molecules, and
others display hydrophobic properties due to the presence of reduced
hydrocarbon fragments. Many enzymes are responsible for the bioconversion of
xenobiotics. Some of these enzymes are listed in Table 2.14.
Table 2.14: Enzymes responsible for the microbial degradation of
pesticides.
Enzyme
Target pesticide
Organophosphates
Esterases
Phenyl carbamates
Dithioates
Lyases
Organophosphates
Organophosphates
Phosphatases
Dithioates
Malathion
Acylamidases
Phenylamide
Chlorinated phenols
Oxygenases
2, 4- D
HCH
DDT
Carbamates
Hydrolases
Chlorinated phenols
2, 4- D
53
s- triazines
Acylanilides
Carbaryl
Dehydrogenases
Cytochrome P-450
Dehalogenases
HCH
DDT
DDT
DDT
HCH
Ref: Singh et. al., 1999, Kumar et. al., 1996.
Although several microbes that degrade xenobiotics have been isolated
and identified, only a few genes have been characterised.
Several genes
encoding some of these enzymes have been identified. The catabolic genes
responsible for the xenobiotic degradation are mostly present on chromosomes.
In a few instances, these genes are located on plasmids or transposons. The
presence of catabolic genes on plasmids makes it easier for the manipulation of
the genes. Further, Chakrabarty and Gunsalus (1971) reported that part of the
camphor degradative pathway genes was plasmid- borne in Ps. putida. Dunn
and Gunsalus (1973) isolated a naphthalene degradative plasmid. The first
report on plasmids encoding the degradation of synthetic pesticides came for
the herbicides 2,4- D and MCPA (Kumar et. al., 1996). Some of the important
plasmids involved in microbial degradation of pesticides is given in Table 2.15.
Table 2.15: Plasmids encoding pesticide degradation.
Pesticide
2,4-D
Plasmid/ fragment
Organism
tfd plasmid
Alcaligenes paradoxus
pJP4
Alcaligenes eutrophus
pRC10
Flavobacterium sp.
pJP1-6
Alcaligenes eutrophus
pEML159
pTFD41
2,4,5- T
pUSI
- HCH
pKJS32
Alcaligenes sp.
Comamonas
Pseudomonas cepacia PT88
Pseudomonas putida DSM549
54
pKTY320:Tn5
Organophosphate
pCMS1
4.3kb plasmid
Pseudomonas putida PpY101
Pseudomonas diminuta MG
Flavobacterium sp. ATCC 27551
Flavobacterium sp.
Carbamate
pSMB2
Carbofuran
mcd, Scal-Clal
Atrazine
TE1
Rhodococcus sps.
pJP4
Alcaligenes eutrophus
MCPA
pRC10
Achromobacter sp. WM111
Flavobacterium sp.
Pseudomonas aeruginosa
Kelthane
pBS3
PCP
80 to 100kb plasmid
Flavobacterium sp.
Bromacil
60 to 100kb plasmid
Pseudomonas sp. strain 50235
Ref: Kumar et. al., 1996
Some of the identified genes involved in the degradation of
nitroaromatics are listed in Table 2.16.
Table 2.16: Identified genes involved in the degradation of nitroaromatics
Target
compound
Enzyme
Encoding
Microroganism
gene
of origin
Monoxygenase
ntnMA
Nitrobenzyl alcohol
ntnD
dehydrogenase
4- nitrotoluene
Nitrobenzaldehyde
ntnC
dehydrogenase
Nitrobenzoate reductase
Pseudomonas
sp. TW3
pnbA
Hydroxylaminobenzoate
2,4dinitrotoluene
lyase
pnbB
Dioxygenase
dntA
Monooxygenase
dntB
Dioxygenase
dntD
Isomerase/hydrolase
dntG
Dehydrogenase
dntE
Burkholderia
cepacia R34
55
2,4,6trinitrophenol
Hydride transferase
npdI
Rhodococcus
Hydride transferase
npdG
erythropolis HL
NADPH reductase
npdC
PM- 1
Reductase
xenB
Ps.fluorescens
2,4,6trinitrotoluene
Source: Eyers et. al., 2004.
Many catabolic genes involved in the catabolism of aromatic compounds
have been cloned and identified from culturable bacteria. Several approaches
such as shotgun cloning by using indigo formation, clearing zone formation or
meta-cleavage as screening methods for cloning (Widada et. al., 2002). The
plasmid- borne parathion hydrolase genes, termed the opd genes, from
Flavobacterium sp. was cloned into Streptomyces lividans, E. coli and insect
cells (Kumar et. al., 1996). The genes encoding the oxygenase component of
the 2,4,5- T oxygenase complex, tft A and tft B from Pseudomonas cepacia
AC1100 were cloned and sequenced (Danganan et. al., 1994). Tomasek and
Karns
(1989)
cloned
the
methyl
carbamate
hydrolysing
gene
from
Achromobacter into Ps. putida. Zylstra et. al. (1989) have reported the cloning
of the toluene dioxygenase gene from Ps. putida and expressed in E. coli. The
haloalkane dehalogenase (dhaA) gene from Rhodococcus rhodochrous NCIMB
13064 was cloned and sequenced (Kulakova et. al., 1997). Gene encoding DL2-haloacid dehalogenase has been cloned from Burkholderia cepacia and
overexpressed in E. coli (Ohkouchi et. al., 2000). Radice et. al. (2007) isolated
an Arthrobacter strain, able to utilize 4-chlorobenzoic acid as the sole carbon
and energy source. The dehalogenase encoding genes (fcb) were sequenced,
cloned and successfully expressed in the heterologous host Pseudomonas
putida PaW340 and P. putida KT2442. Nagata et. al. (1993) have cloned and
sequenced a dehalogenase gene from Pseudomonas paucimobilis involved in
the degradation of - hexachlorocyclohexane. Nardi-Dei et. al. (1997) cloned a
DL-2-haloacid dehalogenase gene from Pseudomonas sp. Jesenska et. al.
(2002) reported the cloning and expression of the haloalkane dehalogenase
gene dhmA from Mycobacterium avium N85. Neumann et. al. (1998) cloned
56
and
sequenced
tetrachloroethene
dehalogenase
pceA
gene
from
Dehalospirillum multivorans and expressed the gene in Escherichia coli.
Genetically constructed strains appear to have some promise for the
cleanup of industrial sewage, a step required for averting environmental
pollution. Successful developments in constructing strains able to degrade
persistent pollutants have brought to the forefront the problems of stability and
resistance of such strains. Therefore, at present, one of the most urgent
problems in this field, is the establishment of the factors which control the
stability of a plasmid-carrying microbial population and the investigation of
possibilities of effective use of constructed strains under natural conditions.
2.17 Conclusions
Though DDT, on its introduction during the second half of World War II
proved effective in controlling vectors spreading typhus and malaria, the
questions on its effects on ecology and human health subsequently led to its
ban for agricultural purposes. The persistence and recalcitrance of the pesticide
in the environment led to its entry to the food chain, leading to bioaccumulation
and biomagnification. The debate over DDT during the 1970s led to its ban. But
as no other alternative to control malaria was available, it was reintroduced
during 2006 by WHO in countries which have failed to combat malaria. Hence, it
is up to the countries to decide to use DDT for public health purposes. 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. Judicious use of this pesticide not only helps to combat
malaria, but also saves our environment.
57
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