DDT and its derivatives (EHC 9, 1979)

INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

ENVIRONMENTAL HEALTH CRITERIA 9

DDT and its Derivatives

This report contains the collective views of an international group of
experts and does not necessarily represent the decisions or the stated
policy of either the World Health Organization or the United Nations
Environment Programme

Published under the joint sponsorship of
the United Nations Environment Programme
and the World Health Organization

World Health Organization
Geneva, 1979

ISBN 92 4 154069 9

^(c) World Health Organization 1979

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CONTENTS

ENVIRONMENTAL HEALTH CRITERIA FOR DDT AND ITS DERIVATIVES

1. SUMMARY AND RECOMMENDATIONS FOR FURTHER STUDIES

1.1. Summary
1.1.1. Properties and analytical methods
1.1.2. Production and uses
1.1.3. Environmental concentrations and exposures
1.1.4. Metabolism
1.1.5. Experimental studies of the effects of DDT
1.1.6. Clinical and epidemiological studies on the effects
of DDT
1.1.7. Dosage-effect relationships
1.1.8. Evaluation of risk
1.2. Recommendations for further studies
1.2.1. Fate in the environment
1.2.2. Monitoring of exposure and effects
1.2.3. Carcinogenicity
1.2.4. Mutagenicity

2. PROPERTIES AND ANALYTICAL METHODS

2.1. Physical and chemical properties of DDT and certain related
compounds
2.1.1. Properties of DDT
2.1.2. Properties of DDT analogues
2.1.3. Formulations of commercial or technical DDT
2.2. Analytical procedures
2.2.1. Statistical criteria for assessing analytical
methods
2.2.2. Limit of analytical detection
2.2.3. Confirmation of the identity of trace residues of
DDT-type compounds
2.2.4. Sampling and extraction
2.2.5. Clean-up procedures
2.2.6. Quantification
2.2.6.1 Determination of DDT-type compounds
2.2.6.2 Determination of p,p'-DDA in urine
2.2.6.3 Method of reporting results
2.2.7. Validation of analytical methods for DDT-type
compounds
2.2.8. Analytical methods for the evaluation of the
biochemical effects of p,p'-DDT and its
analogues

3. SOURCES OF ENVIRONMENTAL POLLUTION

3.1. Discovery and introduction
3.2. Production and use
3.3. Changing patterns of use

4. ENVIRONMENTAL TRANSPORT AND DISTRIBUTION

4.1. Local drift in air
4.2. Distant drift in air
4.3. Distribution in water
4.4. Bioaccumulation of DDT and its degradation in the
environment

5. ENVIRONMENTAL EXPOSURE LEVELS

5.1. Exposure of the general population
5.1.1. DDT in air
5.1.2. DDT in water
5.1.3. DDT in food
5.1.4. Miscellaneous sources
5.1.5. Relative importance of different sources
5.2. Exposure of infants and young children
5.3. Occupational exposure

6. METABOLISM OF DDT

6.1. Uptake
6.1.1. Uptake by inhalation
6.1.2. Uptake from the gastrointestinal tract
6.1.3. Uptake from the skin
6.2. Distribution and storage
6.2.1. Human studies
6.2.1.1 Studies of volunteers
6.2.1.2 Studies of occupationally exposed workers
6.2.1.3 Studies of the general population
6.2.2. Animal studies
6.3. Elimination
6.3.1. Human studies
6.3.1.1 Studies of volunteers
6.3.1.2 Studies of occupationally exposed workers
6.3.1.3 Studies of the general population
6.3.2. Animal studies
6.4. Biotransformation

7. EXPERIMENTAL STUDIES ON THE EFFECTS OF DDT

7.1. Animal studies
7.1.1. Haemopoietic system and immunology

7.1.2. Nervous system and behaviour
7.1.2.1 Cause of death
7.1.2.2 Treatment of poisoning in animals
7.1.3. Renal system
7.1.4. Gastrointestinal tract, liver, and enzymes
7.1.4.1 Liver
7.1.4.2 Microsomal enzymes of the liver
7.1.4.3 Enzymes of intermediary metabolism
7.1.5. Cardiovascular system
7.1.6. Respiratory system
7.1.7. Reproductive system
7.1.8. Endocrine organs
7.1.9. Carcinogenicity
7.1.10. Mutagenicity
7.1.11. Teratogenicity
7.2. Acquisition of tolerance to DDT
7.3. Factors influencing DDT toxicity
7.3.1. Dosage-effect
7.3.1.1 Dosage-effect of DDT
7.3.1.2 Dosage-effect of metabolites and
o,p'-DDT
7.3.2. Age and sex
7.3.3. Nutrition
7.3.4. Species
7.3.5. Other factors
7.4. Human studies

8. EFFECTS OF DDT ON MAN: EPIDEMIOLOGICAL AND CLINICAL STUDIES

8.1. Retrospective studies of DDT-exposed populations
8.1.1. Epidemiological surveillance of persons
occupationally exposed to DDT
8.1.2. Epidemiology of DDT poisoning in the general
population: accidents and suicides
8.1.3. Epidemiology of DDT poisoning in infants and young
children
8.2. Clinical and epidemiological studies of the effects of DDT
on specific organs and systems
8.2.1. Haemopoietic system and immunology
8.2.2. Nervous system
8.2.3. Renal system
8.2.4. Gastrointestinal system
8.2.5. Liver
8.2.5.1 Liver enzymes
8.2.5.2 Other biochemical observations
8.2.6. Cardiovascular system
8.2.7. Reproduction
8.2.8. Endocrine organs

8.2.9. Carcinogenicity
8.2.10. Mutagenicity
8.3. Factors influencing DDT toxicity
8.4. Treatment of poisoning in man

9. EVALUATION OF HEALTH RISKS TO MAN FROM EXPOSURE TO DDT AND
RELATED COMPOUNDS

9.1. Relative contributions of food, water, air, and
miscellaneous sources to total intake
9.1.1. Adult members of the general population
9.1.2. Infants and children
9.1.3. Occupational groups
9.2. Effects of exposure
9.3. Carcinogenicity and mutagenicity
9.4. Effects on microsomal enzymes
9.5. Reproduction and teratogenicity
9.6. Immunosuppression
9.7. Nutritional effects and other factors
9.8. Dosage-effect relationships
9.9. Recommendations on levels of exposure

REFERENCES

ANNEX

REFERENCES

NOTE TO READERS OF THE CRITERIA DOCUMENTS

While every effort has been made to present information in the
criteria documents as accurately as possible without unduly delaying
their publication, mistakes might have occurred and are likely to
occur in the future. In the interest of all users of the environmental
health criteria documents, readers are kindly requested to communicate
any errors found to the Division of Environmental Health, World Health
Organization, Geneva, Switzerland, in order that they may be included
in corrigenda which will appear in subsequent volumes.

In addition, experts in any particular field dealt with in the
criteria documents are kindly requested to make available to the WHO
Secretariat any important published information that may have
inadvertently been omitted and which may change the evaluation of
health risks from exposure to the environmental agent under
examination, so that the information may be considered in the event of
updating and re-evaluation of the conclusions contained in the
criteria documents.

WHO TASK GROUP ON ENVIRONMENTAL HEALTH CRITERIA FOR DDT AND ITS
DERIVATIVES

Participants^a

Members

Dr A. Curley, Toxic Effects Branch, Environmental Toxicology Division,
EPA Health Effects Research Laboratory, Research Triangle Park,
NC, USA

Dr L. Fishbein, National Center for Toxicological Research (US Food &
Drug Administration), Jefferson, AR, USA (Rapporteur)

Dr K. Gheorghiev, Department of Toxicology, Institute of Hygiene &
Occupational Health, Sofia, Bulgaria

Professor F. Korte, Association for Radiation & Environmental
Research, Neuherberg, Munich, Federal Republic of Germany

Professor B. Paccagnella, Second Institute of Hygiene (University of
Padua) Verona, Italy (Chairman)

Professor M. Roberfroid, Department of Biochemistry, Catholic
University of Louvain, Brussels, Belgium

Dr Y. Shirasu, Institute of Environmental Toxicology, Tokyo, Japan

Dr E. M. B. Smith, Department of Health & Social Security, London,
England

Dr D.C. Villeneuve, Biochemical Toxicology Section, Environmental
Health Directorate, Department of National Health & Welfare,
Ottawa, Ontario, Canada (Vice-Chairman)

Representatives of other organizations

Dr M. Stilon de Piro, Occupational Safety & Health Branch,
International Labour Office, Geneva, Switzerland

Mrs M-Th. van der Venne, Health & Safety Directorate, Commission of
the European Communities, Luxembourg

^a Invited but unable to attend:

Secretariat

Dr J. F. Copplestone, Medical Officer, Pesticide Development & Safe
Use, Division of Vector Biology & Control, World Health
Organization, Geneva, Switzerland

Dr Y. Hasegawa, Medical Officer, Control of Environmental Pollution &
Hazards, Division of Environmental Health, World Health
Organization, Geneva, Switzerland

Professor W. J. Hayes, Jr, Department of Biochemistry, Vanderbilt
University, Nashville, TE, USA (Temporary Adviser)

Dr M. Vandekar, Medical Officer Pesticide Development & Safe Use,
Division of Vector Biology & Control, World Health Organization,
Geneva (Secretary)

Dr V. B. Vouk, Chief, Control of Environmental Pollution & Hazards,
Division of Environmental Health, World Health Organization,
Geneva

Dr S. Jensen, Swedish Environmental Analytical Laboratory,
Wallenberg Laboratory, Stockholm, Sweden

Professor R. Truhaut, Toxicological Research Centre, René
Descartes University, Paris, France

ENVIRONMENTAL HEALTH CRITERIA FOR DDT AND ITS DERIVATIVES

A WHO Task Group on Environmental Health Criteria for DDT and its
Derivatives met in Geneva from 8-14 November 1977. Dr V. B. Vouk,
Chief of the Control of Environmental Pollution and Hazards Unit
opened the meeting on behalf of the Director-General. The Task Group
reviewed and revised the second draft criteria document and made an
evaluation of the health risks from exposure to DDT and its
derivatives.

Dr W. J. Hayes, Jr and Dr J. Robinson, Sittingbourne Research
Centre, Kent, England, assisted the Secretariat in preparing the first
and second drafts of the DDT criteria document. Comments on which the
second draft was based were received from the national focal points
for the WHO Environmental Health Criteria Programme in Australia,
Belgium, Canada, Finland, France, Greece, Israel, New Zealand,
Pakistan, and USA and from the International Agency for Research on
Cancer (IARC), the International Labour Office (ILO), the
International Union of Biological Sciences (IUBS), the International
Union of Pure and Applied Chemistry (IUPAC), the United Nations
Industrial Development Organization (UNIDO), and from the United
Nations Environmental Programme International Register of Potentially
Toxic Chemicals.

Comments were also received from Dr V. Benes, Czechoslovakia,
Dr S. Gabor, Romania, and Dr P.M. Newberne, USA.

Two subgroups reviewed the major part of the second draft
(sections 2 to 6 and 7 to 8, respectively) and their comments were
accepted as those of the whole group. Sections 1 and 9 were redrafted
and approved at the plenary sessions.

The collaboration of these national institutions, international
organizations, WHO collaborating centres, and individual experts is
gratefully acknowledged. Without their assistance this document would
not have been completed. The Secretariat wishes to thank, in
particular, Dr W. J. Hayes, Jr for his help in all phases of
preparation of the document.

This document is based primarily on original publications listed
in the reference section. However, several comprehensive reviews on
the health effects of DDT have also been used including publications
by the US Environmental Protection Agency (1975), Müller (1959), and
Mrak (1969).

Although the ecological aspects of DDT, including its possible
accumulation in some components of the food chain, its metabolism in
microorganisms and plants, as well as its effects on terrestrial and
aquatic ecosystems are, no doubt, of great interest and importance,

this document is concerned mainly with the discussion of its
metabolism and effects in experimental animals and man that have
direct implications for human health.

Details of the WHO Environmental Health Criteria Programme
including some of the terms frequently used in the documents may be
found in the general introduction to the Environmental Health Criteria
Programme published together with the environmental health criteria
document on mercury (Environmental Health Criteria 1 -- Mercury, World
Health Organization, 1976), now also available as a reprint.

1. SUMMARY AND RECOMMENDATIONS FOR FURTHER STUDIES

1.1 Summary

1.1.1 Properties and analytical methods

DDT which is an acronym for dichlorodiphenyltrichloroethane is the
prototype of broad action, persistent insecticides. It is stable under
most environmental conditions and is resistant to complete breakdown
by the enzymes present in soil microorganisms and higher organisms.
Some of its metabolites, notably 1,1'-(2,2-dichlor-ethenylidene)-
bis[4-chlorobenzene] (DDE), have a stability equal to, or greater than
that of the parent compound. The persistence of DDT and DDE in the
environment is mainly due to the fact that they are soluble in fat and
virtually insoluble in water.

Two techniques have played a major role in the quantitative
analysis of DDT-type compounds. The original Schechter-Haller
colorimetric method introduced in 1945 was modified in 1953 making it
possible to measure both DDT and DDE in the same sample. A second more
reliable and versatile method for the simultaneous analysis of DDT,
DDE and a number of other organochlorine insecticides began to be used
extensively in about 1962. This consisted of gas-liquid chromatography
with destructive and non-destructive detector systems using
multicolumns for the separation of mixtures. Both methods require
understanding and care in the selection, extraction, clean-up, and
subsequent analysis of samples. Later, gas-liquid chromatography was
combined with mass spectrometry, which added a dimension of mass for
each component of a mixture and provided a more reliable technique for
confirmatory analyses.

Analytical methods, their execution, and the reported results have
not been satisfactory in a number of papers. However, good agreement
has been achieved by analysing paired aliquots by the colorimetric and
gas chromatographic method. In the majority of cases, analytical
errors involving human samples have been small compared with the real
differences, either between individual samples or between groups of
samples drawn from populations with substantially different histories
of exposure. This situation is different with environmental samples
where misinterpretation can occur more often.

1.1.2 Production and uses

Synthesis of DDT was reported in 1874 but its effectiveness as an
insecticide was not discovered until 1939. Because of limited
supplies, most of the compound produced in the world was devoted first
to protection of military areas and personnel, mainly against malaria,
typhus, and certain other vectorborne diseases. Even in 1944, only
4366 tonnes of DDT were produced in the United States of America. The
following year, production reached 15 079 tonnes and, on 31 August

1945, DDT was released for commercial sale. Widespread agricultural
use dates from 1946 in the USA and slightly later in most other
countries.

In the USA, use increased until 1959 (35 771 tonnes) and then
declined gradually so that only 13 724 tonnes were used in 1969.
However, because of the export market, production continued to
increase until 1963 (81 154 tonnes) and then this too gradually
decreased. Unfortunately, there does not appear to be a continuous
record of world production of DDT but according to figures supplied to
the Organization for Economic Cooperation and Development (OECD),
worldwide production in 1974 was 60 000 tonnes. It is known that DDT
has been manufactured in many parts of the world including the
developing countries. However, at present there is only one factory in
the USA, one in France, and one in India.

The ban on the use of DDT and certain other organochlorine
insecticides in Sweden from 1 January 1970 was based on a number of
ecological considerations. More recently a number of other developed
countries have restricted or banned the use of DDT except when it is
needed for the protection of health. However, DDT is still used
extensively for both agriculture and vector control in some tropical
countries. If DDT were not used, vast populations would again be
condemned to the ravages of endemic and epidemic malaria. Substitution
of malathion or propoxur for DDT would increase the cost of malaria
control by approximately 3.4- or 8.5-fold, respectively, and these
increases could not be supported by some countries without decreasing
the coverage of their control programmes.

1.1.3 Environmental concentrations and exposures

When sprayed, some DDT always fails to adhere to the target for
which it is intended and drifts away. Vaporization from treated fields
can be detected for more than six months after application. Most of it
settles in the same area with an almost straight-line, inverse
relationship to the logarithm of the distance from the source.
However, some drift is worldwide. Traces of DDT have been recovered
from dust known to have drifted over 1000 km and in water melted from
Antarctic snow. With rare exceptions, the concentration of DDT in air
in nonagricultural areas has been in the range of <1 to 2.36 × 10^-6 ng/m³.
In agricultural communities, concentrations have ranged from 1 to 22 ×
10^-6 mg/m³. In communities with anti-mosquito fogging programmes,
concentrations of DDT may be much higher, 8.5 × 10^-3 mg/m³ being
the highest level recorded.

Although very difficult to measure, concentrations of DDT in
rainwater have usually been of the same order of magnitude
(1.8 × 10^-5 to 6.6 × 10^-5 mg/litre) in both agricultural areas and
very remote nonagricultural areas, suggesting that the compound is

rather evenly distributed in the air. Presumably because of dust, a
maximum concentration of 4 × 10^-4 mg/litre was found in rainwater in
an urban area. Concentrations of DDT in surface water depend on the
soil as well as on rain. The concentrations in the USA are said to
have reached a peak in 1966 and then dropped sharply. The highest
level ever detected in potable water (2 × 10^-2 mg/litre) was
reported in 1960. In recent years, all concentrations have been
<1 × 10^-3 mg/litre and average concentrations have been similar to
those for rainwater.

Because of drift, DDT concentrations of 0.10-0.90 mg/kg found in
the soil of pastures and other fields not treated with insecticide
were only a little less than those in the soil of cultivated fields
that had been treated with DDT for 10 years or more (0.75 to
2.03 mg/kg). Most of the compound was in the upper 2.5 cm of soil. Due
to evaporation, the total residue of DDT in soils treated for 10 or
more years is of the same order of magnitude as that found soon after
a single application at the same annual rate.

Daily intake of DDT from food has been measured in several
countries. In the USA during 1953-54, average daily intakes of DDT and
of total DDT^a were 0.184 and 0.286 mg/man, respectively, most of
which originated from foods of animal origin. Ten years later,
following restrictions with regard to the application of DDT to
livestock, their barns and forage, and to crops eaten directly by
people, the same investigators found daily intakes of 0.038 and 0.087
mg/man for DDT and total DDT, respectively. The so-called Market
Basket Survey showed a gradual decrease in daily intake of DDT to
0.015 mg/man in 1970. Intake in Canada and the United Kingdom was
slightly less for comparable periods. In many countries of Europe and
in other countries with similar diets, the intake of DDT has been
judged to be about the same because of the similarity of diet and of
measurements of the compound in staple foods and other important items
of the diet. There is a need to measure total intake of DDT with food
in some countries where this has not been estimated. Vegetarians
generally consume less DDT than people who include meat in their diet,
and local practices, including the practices of individual farming
families, may greatly influence the DDT intake of the persons
involved. Extensive use of DDT in the home may contribute moderately
to intake but whether this increased intake is via food is unclear.

With few exceptions, the highest average concentration of DDT in
the air to which workers are exposed (about 7 mg/m³) is that
associated with spraying the inside of houses as for malaria control.
However, concentrations as high as 104 mg/m³ have been reported in

^a Total DDT is a term used to include both DDT and its metabolites
DDE and TDE (DDD).

places where DDT was prepared and packed. Almost all of the DDT in the
air of workplaces is in the form of aerosols. Because of particle size
and other factors, the amount of DDT that workers may inhale is far
less than the amount reaching unclothed portions of their skin. This
is probably important even though DDT is less easily absorbed through
the skin than many other organochlorine insecticides.

More knowledge concerning exposure of workers has been gained from
measurements of the storage of DDT in the body and its excretion than
from environmental measurements. Studies on volunteers have made it
practical to determine intake from either storage or excretion values.
In making these studies on workers, advantage has been taken of groups
employed full time in the manufacture, formulation, or application of
DDT and of others who were in contact with the material
intermittently, sometimes only for a few hours per day and for a few
weeks per year. In the literature, full-time exposure has been
referred to as "heavy" but usually without any intention of implying
that it was excessive or harmful. In fact, improved occupational
safety and health measures have made it possible to reduce the rate of
absorption associated with occupational exposure.

1.1.4 Metabolism

DDT is absorbed after inhalation and ingestion, the latter being
the more important route of absorption. Absorption of large doses is
facilitated by solution in animal or vegetable fat; absorption of
small doses, such as those found in the residues of food, is virtually
complete and is facilitated by the presence of fat in food. Even in
solution, DDT is poorly absorbed through the skin.

Most of the known facts concerning the distribution, storage, and
excretion of DDT have been demonstrated in man as well as in animals.
The compound is stored preferentially in fat, and its storage in
organs and other tissues following repeated intake is proportional to
the neutral fat content of the tissues. However, uptake of DDT by fat
is slow, thus much more is distributed to other tissues following a
single, large dose and much more to adipose tissue following many
small doses. In spite of the affinity of DDT for adipose tissues, most
of the DDT-related compounds in blood are carried by proteins, less
than 1% being carried in the tiny droplets of fat normally present in
the blood.

Following repeated doses, storage in adipose tissue increases
rapidly at first and then more gradually until a steady state is
reached. In each species, the height of the plateau is proportional to
the dosage;^a however, storage is relatively less at higher dosages
because excretion is relatively greater. In man, the time necessary to
reach storage equilibrium is at least one year. There is a gradual
reduction in the amount of DDT stored in the tissues, if exposure to
the compound is discontinued.

Like most species, man converts some DDT to DDE, which is stored
even more avidly than the parent compound. A small amount of 1,1'-(2,2
dichloroethylidene) bis [4-chlorobenzene] (TDE, DDD) an intermediate
in the formation of the main excretory product 2,2-bis(4-chlorophenyl)-
acetic acid (DDA), may also be found in tissues. A number of other
metabolites have been demonstrated in animals but not detected in man.
Technical DDT is more readily excreted and less readily stored than
p,p'-DDT because it contains 15-20% of o,p'-DDT.

DDT dosage-effect relationships have been measured in man by
studying storage and excretion in the general population and in
volunteers. Studies of total diets in the general population revealed
intakes ranging from about 0.02 to 0.20 mg/man per day, in different
subpopulations. In studies on volunteers, dosage was administered
under supervision at the rates of 3.5 and 35 mg/man per day. The
steady-state level of storage in the fat of the volunteers who
received 3.5 mg/man per day was about 50 mg/kg while that of those
receiving 35 mg/man per day was about 300 mg/kg. In recent years, the
concentrations of DDT and of DDT-related compounds stored in adipose
tissues in most populations have averaged <5, and <15 mg/kg,
respectively. Higher values have been found where DDT was used
extensively and without restriction in agriculture or was added
directly to staple foods to control insects. In England and some other
countries where cool weather and a short growing season help to
control insects, the average concentrations of DDT and total DDT
stored in adipose tissues have been <2 and <5 mg/kg, respectively.
In any country, the nonoccupational exposure to DDT and, thus, the
concentrations stored, may vary between subpopulations.

^a The Task Group agreed that, for the purposes of this document,
the term dosage should apply to any rate or ratio involving a
dose, e.g., mg/kg, or (mg/kg)/day.

Most reports of the concentrations of total DDT in the blood of
the general population of different countries lie within the range
0.01 to 0.07 mg/litre. The highest single value reported was
0.336 mg/litre and the highest average value was 0.136 mg/litre. The
concentrations of DDT in the blood and other tissues of the fetus or
newborn are lower than in corresponding tissues of the mother.

Concentrations of DDT in human milk have usually been reported to
be in the range of 0.01 to 0.10 mg/litre with the concentration of DDT
plus its metabolites, especially DDE, about twice as high. However, in
a few countries, average values for total DDT ranging from 1 to
5 mg/litre have been reported, the highest value observed being
12.21 mg/litre.

The average concentration of DDA in the urine of the general
public is 0.014 mg/litre, only slightly less than the lowest
concentration detectable by earlier analytical methods.

Occupational exposure commonly produces average concentrations of
DDT and total DDT stored in fat ranging from 50-175 mg/kg and 100 to
300 mg/kg, respectively. The highest values recorded for DDT and total
DDT in a healthy worker, whose exposure was not measured, were 648 and
1131 mg/kg, respectively. Typical concentrations of DDT and total DDT
in the serum or plasma of workers with substantial exposures have
ranged from 0.14 to 0.57 mg/litre and 0.35 to 1.36 mg/litre,
respectively. The concentration of DDA in the urine of substantially
exposed workers has been in the range of 0.5 to 3.0 mg/litre.
Concentrations of DDT or its derivatives in fat, serum, or urine may
be used to estimate the dose, if exposure has been prolonged and
essentially steady. The ranges of storage and excretion, just
mentioned, were measured in workers who were found to have absorbed a
total dosage ranging from 0.25 to 0.5 (mg/kg)/day.

Animal studies indicate that the concentration in serum most
accurately reflects the concentration in the brain, the critical
tissue. In the rat, a level in the brain of 25 mg/kg is not usually
fatal although higher levels tend to be.

1.1.5 Experimental studies of the effects of DDT

The toxicity of a single dose is affected by the solvent vehicle
and representative median lethal dosage (LD₅₀) values for the rat
are 250 mg/kg, for oral administration in oil, and 250-500 mg/kg or
3000 mg/kg for dermal administration in oil, or powder, respectively.

Large doses of DDT produce vomiting in man and other species that
can vomit and this can modify the amount absorbed.

The main effect of DDT is on the nervous system. All parts, both
central and peripheral, are affected to some degree. In animals,
single or repeated doses can produce hyperexcitability, tremor,
ataxia, and finally epileptiform convulsions. Ataxia may be
demonstrated by functional tests in animals that have received daily
dosages too small to produce noticeable clinical effects. Death is
usually due to respiratory failure at the convulsive stage of
poisoning. In some species, DDT sensitizes the heart to arrhythmia,
which is made worse by epinephrine of endogenous or exogenous origin,
and these animals die in ventricular fibrillation.

It appears that the mechanism of the toxic action of DDT is
associated with its effect on the membranes in the nervous system. In
vitro concentrations as low as 10^-8 mol/litre change the movement
of both sodium and potassium ions through the axonal membrane, and
this movement is involved in the transmission of nervous impulses.
Other evidence of nervous system effects are changes in the
concentrations of 4-(2-amino-1-hydroxyethyl)-1,2-benzenediol
(norepinephrine) and other neurotransmitters in poisoned animals.

Apart from the nervous system, the liver is the only other organ
significantly affected by DDT. Potentially fatal doses of the compound
cause focal necrosis of liver cells in several species. These lesions
heal by autolysis and phagocytic action in animals that survive. A
distinct form of liver cell change reflecting stimulation of
microsomal enzymes is for all practical purposes confined to rodents.
DDT induces microsomal enzymes in all species tested, but only in some
rodents does the endoplasmic reticulum increase so much that the
entire liver cell enlarges and granules that are normally scattered
throughout the cytoplasm are displaced to the margin of the cell.
These changes are accompanied by a moderate increase in fat droplets
some of which become surrounded by whorls of endoplasmic reticulum to
form so-called lipospheres. These characteristic changes have been
observed by electron micrography as early as four days after
administration of DDT, and may have occurred even earlier.

If DDT is fed for long periods at dietary levels ranging from
2 mg/kg upwards for mice or 5 mg/kg upwards for rats, the changes in
the liver progress from hypertrophy, margination, and lipospheres in
isolated, centrolobular hepatocytes to the formation of nodules of
affected cells. The first change has been observed within 4 days of
administration, the earliest time of observation. With administration
of dosages corresponding to those that people may encounter,
changes in the livers of susceptible rodents require the entire
lifetime of the animal to develop fully. At first, the nodules are
microscopic in size, but some may become more than a centimetre in
diameter, particularly in mice, and show almost complete loss of
lobular architecture. The same series of changes can be produced in
rodents by other inducers of microsomal enzymes, including
phenobarbital. Although there is persuasive evidence that these
multinodular tumours of mice associated with changes in the

endoplasmic reticulum are carcinomas, there is equally convincing
evidence that they are not and the views of some highly qualified
pathologists in this matter remain diametrically opposed. More
important than the question of classification is the fact that the
entire continuum of changes from the prompt response in isolated cells
to the eventual formation of tumours is peculiar to some rodents, and
does not occur in other animals in which the endoplasmic reticulum
does not respond morphologically in the same way.

A number of enzymes of intermediate metabolism are either
stimulated or moderately inhibited by toxic doses of DDT; the
possibility that these changes are the result rather than the cause of
poisoning has not been excluded.

Levels of DDT as high as 200 mg/kg of food that do not produce any
sign of poisoning, have not produced any adverse effects on fertility,
gestation, viability, and lactation, and on the health of the progeny
of rats and mice. Reproduction was normal in dogs receiving a dosage
of 10 mg/kg body weight per day, which is approximately equivalent to
a dietary level of 500 mg/kg for this species.

No teratogenic effects of DDT have been observed in multigeneration
studies of reproduction in several animal species.

There is some uncertainty concerning the effects of DDT on the
immune system; where an effect has been observed, it has been of a
probiotic nature.

Except for the weak estrogenic properties of o,p'-DDT, the
endocrine related effects of DDT and its analogues are confined to the
adrenals and even these effects are now considered to be mainly
secondary to microsomal enzyme induction in the liver.

DDT has not been found to be mutagenic in bacterial test systems,
either without or with metabolic activation. The evidence from
mammalian test systems, in vitro and vivo is inconclusive.

No specific antidote for DDT poisoning is known, but sedatives
(especially phenobarbital) and ionic calcium are useful for treating
poisoning in dogs and monkeys. Glucose or other ready sources of
energy are also helpful in treatment.

1.1.6 Clinical and epidemiological studies on the effects of DDT

Mild poisoning was produced in one volunteer who ingested 750 mg
DDT in oil in order to study its effects. All other poisoning of human
subjects by DDT has been the result of accidental or suicidal
ingestion. No systemic poisoning has resulted from occupational
exposure to DDT, but a few workers have developed rashes or irritation

of the eyes, nose, and throat associated with dust. Most of the very
few fatal cases have involved children who drank solutions of the
compound and whose clinical courses were dominated by solvent
poisoning.

Signs of DDT poisoning in man are entirely similar to those
observed in animals. In addition, persons poisoned have experienced a
prickling sensation of the tongue and around the mouth and nose,
reduction of tactile sense, paraesthesia of the extremities, nausea,
dizziness, confusion, headache, malaise, and restlessness. In most
patients, all signs and symptoms (including vomiting) probably
involved the nervous system; a few had temporary jaundice indicating
liver injury. In the majority of survivors, recovery was well advanced
in 24 hours but a few required a week or more. Three men still had
some weakness and ataxia in their hands five weeks after ingesting an
amount estimated to be as high as 20 000 mg of DDT per person.

Stimulation of microsomal enzymes of the liver has resulted from
full-time occupational exposure and from the therapeutic use of DDT in
the treatment of familial, nonhaemolytic, unconjugated jaundice.

The only demonstrated effects of DDT on the general population are
the storage of the compound and some of its derivatives in the tissues
and their excretion in urine and milk. No confirmed ill-effects of DDT
have been reported in babies, even in communities where the highest
concentrations of the compound in human milk have been observed.

Careful investigation of the largest available groups of workers
who have been exposed for as long as 25 years to significantly higher
levels of DDT than the general population, has not revealed any
evidence that DDT causes cancer in man. The total number of people in
the world who have had many years of full-time occupational exposure
to DDT is smaller than might be supposed. This makes the detection of
any effect with a low incidence difficult. While it has been
recognized that some human carcinogens have been detected only after
comparatively long periods of exposure, it is also known that others
(e.g., 2-naphthylamine) have been detected through their occurrence in
high incidence in small groups following exposure for periods of much
less than 25 years.

1.1.7 Dosage-effect relationships

Dosage-effect relationships for DDT in man have been observed in
connection with acute poisoning, excretion, and storage, and the
induction of microsomal enzymes has been observed at a dosage of
0.25 (mg/kg)/day but not at lower dosages. The dosage of 0.25 (mg/kg)/
day to which workers have been exposed for 25 years is of the same
order of magnitude as the dosage that causes an increase in the
incidence of tumours in male mice of a susceptible strain but not
in females of any strain. (See section 1.2.4). This same dosage is

lower than the nonobserved effect levels for rats, dogs, and monkeys
and far less than the dosage at which rats, mice, and dogs continue to
reproduce successfully for generations.

1.1.8 Evaluation of risk

Food represents the major source of intake of DDT for the general
population. The average intake of DDT from all sources is unlikely to
exceed 0.05 (mg/man)/day. Occupational exposure to DDT is mainly
respiratory and dermal. However, much of what is inhaled is deposited
in the upper respiratory tract and subsequently ingested. The effects
of dermal exposure are minimal because the compound is poorly absorbed
through the skin; the excellent safety record, never matched by any
other insecticide used in antimalaria campaigns, other vector control
programmes, and agriculture is due mainly to this fact. The number of
people throughout the world currently engaged in the manufacture of
DDT is small and, wherever safety and health protection measures are
good, occupational exposure of this group is minimal. Formulators and
applicators are also groups that are occupationally exposed to DDT but
there is no evidence to suggest that their intake is significantly
higher than that of workers engaged in the manufacture of DDT.

No adverse effects have been described in man at repeated dosage
of 1.5 (mg/kg)/day. The large number of measurements that have been
made on samples from human populations do not throw any real light on
the question of maximum-tolerated doses or concentrations, apart from
highlighting the fact that the levels found in volunteers and workers
which were higher than those in the general population were not
associated with any adverse effects.

In the light of currently available information, there is no
evidence that DDT is carcinogenic in man. Liver tumours are produced
in mice and possibly in rats by DDT, DDE, and TDE but there is
disagreement on the significance of these tumours. Studies in
in vitro bacterial test systems have not shown any evidence that
either DDT or DDE is mutagenic. The evidence from mammalian test
systems, both in vitro and in vivo, is inconclusive.

DDT induces microsomal mixed function oxidases in many animal
species and causes marked morphological changes in the liver of some
rodents. At the present time, it is very difficult to assess the
biological significance of this effect for men, since the intake by
members of the general population is much lower than the smallest
dally dosage required to produce such an effect in man and animals.

In both man and animals, there, is no indication that DDT affects
reproduction or produces teratogenic effects although it has been
shown to be embryotoxic in high doses.

DDT appears to have a depressant effect on the immune system
although the evidence is by no means conclusive.

Animal studies indicate that nutritional status influences the
toxicity of DDT. In man, nutritional status will have a similar effect
to that in animals. However, the possibility that starvation in man
could precipitate toxic manifestations is regarded as unlikely as the
stored levels do not approach those found in laboratory animals.

The information derived from human exposure is insufficient to
construct a comprehensive picture of the dosage-effect relationships
for man except in connection with storage and excretion of the
compound and its metabolites.

1.2 Recommendations for Further Studies

DDT is the first synthetic pesticide to which many people have
been exposed to a measurable degree for a period of many years. It has
already been the subject of an enormous amount of scientific study,
but of course there is still more to be learned. The following
recommendations are considered to have special implications for human
health. Much other important biomedical research such as the
continuing use of DDT as a tool for studying the nervous system has
been excluded from this document.

1.2.1 Fate in the environment

There is a serious gap in knowledge of the circulation and fate of
DDT and its analogues in the environment as a whole. Because this is
directly connected with the assessment of future exposure pathways for
man, the behaviour and fate of DDT in the environment should be
studied more extensively. Great progress has been made recently in
demonstrating the breakdown of DDT to carbon dioxide and hydrochloric
acid under laboratory conditions similar to those found in the upper
atmosphere. There is a need for further study of this phenomenon in
the laboratory, especially using DDT labelled with ¹⁴C and ³H.
There is an even greater need for seeking quantitative information on
the rate at which photomineralization may occur in nature and the
factors that influence this rate.

1.2.2 Monitoring of exposure and effects

There are fairly accurate estimates of the daily intake of DDT in
several developed countries. In other countries where DDT is most
likely to be used continuously, the daily exposure of the general
population to DDT in food should be monitored, especially if there are
indications that the conditional acceptable daily intake (ADI) of
0.005 (mg/kg)/day might be exceeded. For comparison, monitoring
programmes should be continued in countries where figures are
available for earlier years.

Extensive information is available on the occurrence of DDT and
its metabolites in human fat, blood, and milk. Continued, but limited
monitoring is justified in order to learn the rate at which
concentrations decline following a progressive reduction in the use of
the compound. More extensive monitoring is justified in countries
where base data are not available and where the use of the insecticide
is essential. Particular attention should be given to people with
substantial occupational exposure. If values are found that are not
consistent with the dietary and occupational history of each group,
the cause of the variation should be sought. If values are
unexpectedly high, some improper use may be discovered. If values are
unexpectedly low, some modifying factor such as previously
unrecognized intake of phenobarbital may be revealed.

Clinical studies should be made on any person or group found to
have exceptional levels of storage or excretion in the hope of
learning whether the insecticide has had any influence on their health
or, conversely, to learn whether their nutritional state or the
presence of chronic disease has interacted in any way with storage and
excretion. Obviously such studies must be adequately controlled if the
results are to be of use.

1.2.3 Carcinogenicity

Attention should be turned from the narrow question of the
tumorigenicity of DDT in the liver of mice and rats to the broader
question of the basis for this action of DDT and phenobarbital. A far
wider range of inducers should be studied, keeping in mind that some
inducers may have other important properties related to the initiation
of tumours. Compounds belonging to several classes (e.g., an
organochlorine insecticide, phenobarbital, a pyrethrin, etc.) should
be studied in several species (including one nonrodent) to determine
the dosage-response relationships for: (a) microsomal enzyme activity;
(b) typical morphological changes in the endoplasmic reticulum of
hepatocytes; and (c) liver tumours. These studies together with
continuing epidemiological investigations of the effects of the same
classes of compounds on people should make it easier to extrapolate
from tumorigenesis in animals to the problem of cancer in man.

1.2.4 Mutagenicity

There is satisfactory evidence that DDT is not mutagenic in
bacterial systems, without and with metabolic activation. The evidence
derived from mammalian test systems, both in vitro and in vivo is
inconclusive and should be clarified. Methods of mutagenicity testing
are advancing rapidly, and shorter and possibly more sensitive
mammalian tests are becoming available. A fresh evaluation of the
mutagenicity of DDT in animals would facilitate further assessment of
its significance for man.

2. PROPERTIES AND ANALYTICAL METHODS

2.1 Physical and Chemical Properties of DDT and Certain Related
Compounds

2.1.1 Properties of DDT

The term DDT is generally understood throughout the world and
refers to 1,1'-(2,2,2-trichloroethylidene)-bis(4-chlorobenzene)
( p,p'-DDT). The structure of DDT permits several different isomeric
forms, an example of which is 1-chloro-2[2,2,2-trichloro-1-(4-
chlorophenyl)ethyl]benzene ( o,p'-DDT). The term DDT is also applied
to commercial products consisting predominantly of p,p'-DDT together
with some o,p'-DDT and smaller amounts of other compounds. A typical
example of technical DDT had the following composition: p,p'-DDT,
77.1%; o,p'-DDT, 14.9%; p,p'-TDE, 0.3%; o,p'-TDE, 0.1%; p,p'-DDE,
4.0%; o,p'-DDE, 0.1%; and unidentified compounds, 3.5%.

All isomers of the compound DDT are white, crystalline, tasteless,
almost odourless solids with the empirical formula C₁₄H₉Cl₅ and a
relative molecular mass of 354.5. The melting range of p,p'-DDT is
108.5-109.0°C and its vapour pressure is 2.53 × 10^-5 Pa
(1.9 × 10^-7 mm Hg) at 20°C. DDT is soluble in organic solvents as
follows (g/100 ml): benzene, 106; cyclohexanone, 100; chloroform, 96;
petroleum solvents, 4-10; ethanol, 1.5. It is highly insoluble in water.

p,p'-DDT is dehydrochlorinated to form DDE (see Table 1) at
temperatures above the melting point, especially in the presence of
catalysts or light. Solutions in organic solvents are
dehydrochlorinated by alkali or organic bases. Otherwise, DDT
formulations are highly stable. The compound is also relatively
resistant to breakdown by the enzymes found in soil and higher
organisms, and DDE is even more resistant. Under simulated atmospheric
conditions, both DDT and DDE decompose to form carbon dioxide and
hydrochloric acid.

2.1.2 Properties of DDT analogues

The chemical structure of some of the analogues of DDT is shown in
Table 1. The structure of the o,p'- and m,p'-compounds can be inferred
from those of the p,p'-isomers. The table is confined to
compounds that occur in commercial DDT, metabolites formed from them,

Table 1. Structure of p,p'-DDT and its analogues of the form:

(many of the compounds also exist as o,p'-isomers and other isomers)

Name DDT Chemical name R R' R"
and its major
metabolites

DDT 1,1'-(2,2,2-trichloroethylidene)- -Cl -H -CCl₃
bis[4-chlorobenzene]
DDE^a 1,1'-(2,2-dichloroethenylidene)- -Cl None =CCl₂
bis[4-chlorobenzene]
TDE(DDD)^a,b 1,1'-(2,2-dichloroethylidene)- -Cl -H -CHCl₂
bis[4-chlorobenzene]
DDMU^a 1,1'-(2-chloroethenyldene)- -Cl None =CHCl
bis[4-chlorobenzene]-
DDMS^a 1,1'-(2-chtoroethylidene)- -Cl -H -CH₂Cl
bis[4-chlorobenzene]
DDNU^a 1,1'-bis(4-chlorophenyl)ethylene -Cl None =CH₂
DDOH^a 2,2-bis(4-chlorophenyl)ethanol -Cl -H -CH₂OH
DDA^a 2,2-bis(4-chlorophenyl)- -Cl -H -C(O)OH
acetic acid

Table 1 (Cont'd)

Name DDT Chemical name R R' R"
and its major
metabolites

Some related insecticides

NO₂
Bulan^(R) 2-nitro-1,1 -bis- -Cl -H '
(4-chlorophenyl)butane -CHC₂H₅

NO₂
Prolan^(R) 2-nitro-1,1-bis- -Cl -H '
(4-chlorophenylpropane -CHCH₂

DMC 4-chloro-alpha[-(4-chlorophenyl)- -Cl -OH -CH₃
alpha-(methyl)benzenemethanol

dicocol 4-chloro-alpha-(4-chlorophenyl)-alpha- -Cl -OH -CCl₃
(Kelthane^(R)) (trichloromethyl)benzenemethanol

chlorobenzilate^c ethyl 4-chloro-alpha-(4-chlorophenyl)- -Cl -OH -C(O)OC₂H₅
alpha-hydroxybenzeneacetate

chloropropopylat^c 1-methylethyl 4-chloro-alpha- -Cl -OH -C(O)OCH(CH₃)₂
(4-chlorophenyl)-aplha-hydroxy-
benzeneacetate

methoxychlor^c 1,1'-(2,2,2-trichloroethylidene)- -OCH₃ -H -CCl₃
bis[4-methoxybenzene]

Table 1 (Cont'd)

Name DDT Chemical name R R' R"
and its major
metabolites

Perthane^(R) 1,1'-(2,2-dichloroethylidene)- -C₂H₅ -H -CHCl₂
bis[4-ethylbenzene]

DFDT 1,1'-(2,2,2-trichloroethylidene)- -F -H -CCl₃
bis[4-fluorobenzene]

^a Recognized metabolite of DDT in the rat.
^b As an insecticide, this compound has the ISO approved name of TDE, and it has been sold
under the name Rothane(R); in metabolic studies the same compound has been referred to as
DDD; as a drug, it is called mitotane.
^c Common name approved by the International Organization for Standardization (ISO).

and analogues that have had some use as insecticides. It must be
emphasized that even the commercially-available insecticidal analogues
have strikingly different properties. Especially remarkable is the
slow metabolism and marked storage of DDT and its metabolite DDE and
the rapid metabolism and negligible storage of methoxychlor.

No attempt has been made to include in Table 1 the wide range of
compounds that have been synthesized and studied in connexion with
structure-activity relationships, often with the hope of emphasizing
the good properties of DDT and reducing its undesirable properties.
For a more extensive consideration of analogues, see Metcalf (1955).

The formation of metabolites is considered in section 6.4.

2.1.3 Formulations of commercial or technical DDT

Technical DDT has been formulated in almost every conceivable form
including solutions in xylene or petroleum distillates, emulsifiable
concentrates, water-wettable powders, granules, aerosols, smoke
candles, charges for vaporizers, and lotions. Aerosols and other
household formulations are often combined with synergized pyrethrins.

When used as a drug, DDT is called clofenotane (INN) or Dicophane
(British Pharmacopoeia), Klorfenoton (Swedish Pharmacopoeia),
Chlorophenothane (United States Pharmacopoeia). For research or
reference it has been designated OMS 0016 and Ent. 1,506. DDT has been
sold under a variety of tradenames, including: Anofex(R), Cezarex(R),
Dinocide(R), Gesarol(R), Guesapon(R), Guesarol(R), Gyron(R),
Ixodex(R), Neocid(R), Neocidol(R), and Zerdane(R).

2.2 Analytical Procedures

Analytical procedures for determining residues of DDT-type
compounds in environmental samples involve several steps including
collection and extraction of the DDT-type compounds; removal of
coextractives by appropriate clean-up methods; and quantification of
p,p'-DDT and its analogues by a suitable technique. Each of these
major steps is discussed later. It is appropriate, however, first to
outline briefly the statistical criteria used to assess analytical
methods, the estimation of the lower limit of detection of a method,
and the procedure for confirming the chemical identities of the
components measured.

2.2.1 Statistical criteria for assessing analytical methods

The overall reliability of an analytical method can be assessed
using two criteria, namely, reproducibility and systematic error (or
bias). Reproducibility is both conceptually and practically the
simpler criterion; it may be defined as "the quantitative expression
of the random error associated with operators working in different

laboratories, each obtaining single results on identical test material
when applying the same method" (Institute of Petroleum, 1968). It may
be quantitatively specified in various ways, and it is important to
pay attention to the statistic (range standards, deviation, etc.) used
in a particular study to represent the random error of an analytical
method. If the results are normally distributed then the most
efficient statistic measuring reproducibility is the standard
deviation of a set of results (Nalimov, 1963; Youden & Steiner, 1975;
Davies & Goldsmith, 1976). Care should always be taken to ensure that
a particular statistic or statistical technique is appropriate for a
given set of results, by, for example, testing for outliers or, if
there are sufficient results, examining the distribution of the
results, before characterizing the reproducibility of an analytical
method.

There are two subdivisions of the reproducibility criterion,
namely, replication i.e., two or more results, obtained by the same
operator in a given laboratory using the same apparatus for successive
determinations on identical test material, within a short period of
time on the same day; and repeatability i.e., a quantitative
expression of the random error associated in the long run with a
single operator in a given laboratory obtaining successive results
with the same apparatus under constant operating conditions on
identical test material (Institute of Petroleum, 1968).
Reproducibility is, in turn, a subdivision of the random error in the
analysis of identical test material in different laboratories using
different techniques or variations of a particular method. Examples of
the assessment of the random errors found in the determination of
p,p'-DDT and related compounds are given below.

The systematic error of a method is the deviation of the
experimental results from the "true" values; such systematic error
causes the differences between the nominal value and the
experimentally-determined values to have predominantly the same sign
(as opposed to the random errors, where the results are equally likely
to be greater than or less than the true mean). Nominal or "true"
values are available only in the case of fortified (spiked) samples,
but whether such fortified samples are representative of actual
(environmentally incurred) contamination is open to doubt in the case
of some types of material. A rapid nonparametric test of systematic
error can be made using the sign-test or the Wilcoxan signed ranks
test (Conover, 1971).

It is common practice to calculate the "recovery" factor for an
analytical method, i.e., the ratio of the mean observed value to the
nominal value (usually expressed as a percentage), but the statistical
significance of the "recovery" factor should always be assessed. The
ratio of the mean deviation to the standard error of the mean

deviation is an appropriate method of testing the null-hypothesis that
the difference between the nominal value and the observed mean is not
statistically significant.

The term "total error" has been proposed for a function
incorporating both the systematic error and the reproducibility
(McFarren et al., 1970), and these authors also suggested three
classes of total error corresponding to excellent methods, acceptable
methods, and methods that are judged unacceptable. It is pertinent
that McFarren et al. concluded that the total errors in one study of
the determination of DDT-type compounds in water were unacceptable
(>50% for p,p'-DDT, and p,p'-DDE, 24.0-53.6% for o,p'-DDT).

2.2.2 Limit of analytical detection

All analytical determinations have a lower limit corresponding to
that quantity (Delta g) of p,p'-DDT (or a related compound) which
produces a response (Delta r) that cannot be distinguished from
response (Delta r) produced when no p,p'-DDT is present. The
response Delta r is generated by the materials, reagents, and
instruments, used in the procedure, for example, the small voltage
generated by electronic equipment, or the small absorption in a
spectrophotometric method. These responses are known as "blank" or
"noise", and their size depends on the presence of interfering
components in the test material, the purity of the reagents, the
cleanliness of the apparatus, and the design of electronic equipment
(amplifiers, etc.). The concept of limit of detection is a statistical
one and is related to the random variation of the response generated
by a blank or control (Sutherland, 1965; Skogerboe & Grant, 1970;
Kaiser, 1973). Currie (1968) has defined three limiting levels for use
in analytical chemistry: "the net signal level" above which an
observed signal may be reliably recognized; "the true net signal
level" which may, a priori, be expected to lead to detection; and
"the quantifiable level" at which the measurement precision is
sufficient for the quantity present to be estimated satisfactorily. In
many types of sample, the limit of detection is of rather academic
interest as the concentrations of DDT-type compounds in samples are an
order of magnitude greater than the limit of detection of a sensitive
detector such as the electron-capture detector. However, in analyses
of air and drinking-water, for example, it may be necessary to
ascertain the limit of detection by a suitable statistical procedure.

Lower limits of detectability (ng.g^-1) suggested in the US EPA
Manual of Analytical Methods (Thompson, 1974) for DDT-type compounds
using gas-liquid chromatography are:

o,p'-DDE Adipose tissue, 10; serum, 1
p,p'-DDE Adipose tissue, 20; serum, 1
o,p'-DDT)
p,p'-TDE) Adipose tissue, 20; serum, 2
p,p'-DDT)

2.2.3 Confirmation of the identity of trace residues of DDT-type
compounds

Confirmation of the identity of p,p'-DDT and related compounds
in many types of environmental samples is not easy when the apparent
amounts present are in the microgram and submicrogram range, since the
classical procedures for the identification of organic compounds
require the use of milligrams of the purified compound. A definition
of chemical identity appropriate to trace analysis has been proposed
(Robinson et al., 1966), a definition that may need revision in
relation to the combined use of gas-liquid chromatography and mass-
spectrometry. Chemical derivatization techniques for DDT-type
compounds have been reviewed by Cochrane & Chau (1971). Other
techniques, that are used routinely in the confirmation of the
identity of DDT-type compounds include: determination of gas-liquid
chromatographic retention times using polar and nonpolar stationary
phases; thin-layer chromatography; paper chromatography; p-values;
infrared microtechniques; carbon skeleton chromatography; conversion
into dichlorobenzophenones; nuclear magnetic resonance; and X-ray
diffraction. Tables of the relative retention times (aldrin = 1.00) of
DDT-type compounds using nine liquid phases (Thompson et al., 1975)
and three mixed liquid phases (Suzuki et al., 1975); gas-liquid
chromatographic retention times for DDT-type compounds are also
summarized by Yermakov (1972). Relative thin-layer chromatographic
R^f values ( p,p'-DDE = 1.00) on alumina using three solvent systems
were reported by Thomas et al. (1968). Extraction p-values for
DDT-type compounds using seven solvent systems are given in the US EPA
Manual (Thompson 1974). Identification of DDT and its metabolites by a
microinfrared technique has been studied by Sierwiski & Helrich (1967)
and reference infrared spectra have been published (Chen et al.,
1972). Asai et al. (1971) used carbon skeleton chromatography to
differentiate polychlorinated biphenyls from DDT-type compounds. The
conversion of DDT-type compounds into the corresponding benzophenones
may be used to identify them in the presence of polychlorinated
biphenyls (Miles, 1972), but this technique has its drawbacks as
p,p'-DDT, p,p'-TDE and p,p'-DDE all give the same p,p'-
dichlorobenzophenone. The use of high resolution nuclear magnetic
resonance spectroscopy (with a time averaging computer to increase
sensitivity), for the confirmation of the presence of p,p'-DDT and
p,p'-DDE in human adipose tissue (total concentration about
13 mg/kg) was studied by Biros (1970).

The combination of gas-liquid chromatography with mass-
spectrometry is a powerful tool for the confirmation of identity of
trace residues of DDT-type compounds. For example, Gordon & Frigerio
(1972) used mass fragmentography and claimed identification of 10 pg
p,p'-DDT; Schaeffer (1974) identified DDT-type compounds in fish
using a fast scan mass-spectrometer to give a multiple mass spectrum
for each of the overlapping peaks.

2.2.4 Sampling and extraction

Before discussing different kinds of samples, it must be noted
that valuable information on many analytical problems may be found in
the US EPA Manual (Thompson, 1974) and the FDA Manual (McMahon &
Sawyer, 1977).

DDT-type compounds may be present in the air in vapour form or
adsorbed on particulate matter. Glass-fibre filters are suitable for
trapping the particulate matter (Stanley et al., 1971; Beyermann &
Eckrich, 1974) and DDT in the vapour form may be trapped using
impingers of the Greenburg-Smith type and a suitable nonvolatile
solvent using ethylene glycol. Miles et al. (1970) reported a trapping
efficiency of more than 99% for DDT. A three-trap system in series
comprising a column containing glass cloth, followed by an impinger
with hexylene glycol, and finally an alumina column was used by
Stanley et al. (1971). They estimated that the collection efficiency
(based on material balance studies) of this system for a mixture of
aerosol and vapour was about 60% for p,p'-DDE, 95% for p,p'-TDE,
and 100% for p,p'-DDT. Beyermann & Eckrich (1974) used glass wool
for the trapping of aerosols, and a stainless-steel net coated with
polyethylene glycol for trapping vapours. Trapping of organochlorine
insecticides in the vapour phase using support-bonded silicones on
various types of Chromosorb was considered to be quantitative by Aue &
Teli (1971): DDT-type compounds were not examined but the trapping of
lindane, aldrin, and heptachlor was considered satisfactory. A cross-
linked polystyrene resin, Chromosorb 102, was used by Thomas & Serber
(1974). These workers reported a collection efficiency of 98% for
o,p'-DDT at a concentration of 15 ng/m³. Herzel & Lahmann (1973)
used silica gel as the support for various liquid phases and concluded
that polyethylene glycol was the best absorbent for DDT-type compounds
in the air. Absolute calibration of the collection efficiency of
aerosols and atmospheres for DDT-related compounds is difficult, but
the studies that have been made indicate that impingers with ethylene
glycol or hexylene glycol are probably the most efficient. The
simplest empirical test of the efficiency of the trapping system is to
use two (or more) impingers in series and analyse the liquid
absorbents from the impingers separately. The ratio of the amounts
present in the first and second impingers should be higher than 10:1.
Detailed instructions for the sampling and analysis of air for
pesticides (including DDT-type compounds) by a method that has been
subjected to interlaboratory study are given in the US EPA Manual
(Thompson, 1974).

For the determination of DDT-type compounds in water an
uncontaminated container may be filled, but if the concentrations of
DDT are likely to be extremely low (as would be expected in most
potable waters), then drawing the water through a suitable trapping
device may he more appropriate; this method also gives a time-weighted
average concentration and can be used in an automated monitoring
procedure.

Benzene was used by Pionke et al. (1968) as the solvent to extract
p,p'-DDT and p,p'-TDE from fortified samples of distilled water
and lake waters. The levels of fortification were high (µg/litre) and
the recoveries were: p,p'-DDT, 96.1% ± 1.02%; p,p'-TDE 97.3% ± 0.89%.
Thus, at concentrations of the order of 0.001 mg/litre, the
benzene extraction method gives excellent recoveries (total errors,
5.9% and 4.5%, respectively). A continuous flow method based
on liquid-liquid partition was developed by Ahling & Jensen (1970);
water is passed through a column containing Chromosorb W coated with
undecane plus a macrogol Carbowax 4000 monostearate. The best
collection efficiencies were found when the two liquid absorbents were
used at concentrations of 10% and 30%, respectively, on the solid
support. At concentrations in the ng/litre range, the optimum recovery
of known amounts of DDT-type compounds added to water were obtained
with 1.5 g coated Chromosorb W per litre of water. Ahnhoff & Josefson
(1974) used continuous flow liquid-liquid partition between water and
cyclohexane (in three extractors in series). The extraction
efficiencies from water containing 5 ng p,p'-TDE or 7.6 ng p,p'-DDT
per litre were greater at a flow rate of 2 litre/h than at
5 litre/h being 90% and 98% for p,p'-DDT, and p,p'-TDE,
respectively, with 92% and 93%, respectively, of the recovered
compounds being found in the first extractor. A method using a solid
adsorbent, a macroreticular resin, XAD-4, has been studied by Musty &
Nickless (1974); at a flow rate of 8 ml/min through a column
containing 2 g XAD-4, satisfactory recoveries of p,p'-DDT and three
related compounds were obtained at concentrations of 2-10 ng-litre.
Carbon is a very efficient adsorber of p,p'-DDT and p,p'-TDE from
water (Rosen & Middleton, 1959), but desorption from charcoal is
difficult; furthermore, the recoveries are not very satisfactory and
this is attributed to chemical changes catalysed by carbon in contact
with water rather than inefficient desorption (Eichelberger &
Lichtenberg, 1971). Another solid adsorbent is polyethylene (Beyermann
& Eckrich, 1973), but in the case of river water variable results were
obtained because of the effects of other solutes. Taylor & Bogacka
(1968) used petroleum ether to extract DDT from water; following a
clean-up with acetonitrile partition and Florisil the overall recovery
(using thin-layer chromatography) was incomplete (about 66%). The
total error of this procedure is unacceptable. Benzene was used as the
extraction solvent by Djatlovitskaja et al. (1972), and a general
review of the determination of organochlorine insecticides (and other
insecticides) in water has been published by Novikova (1973).

The need for scrupulous cleanliness in glassware and purity of
reagents cannot be overstressed in the case of the determination of
DDT-type compounds in air and water as the residues are usually so
small; the use of electronic equipment with a low noise characteristic
and constant checking of the response of detectors are also necessary.

A major difficulty in the determination of organochlorine
insecticides in soils is the initial extraction of the residues
because of a combination of factors, including the wide variation in

soil types. Chiba (1969) emphasized the lack of precision in defining
soil types. In his review article, he concluded that the most
effective solvent systems for extraction of these compounds were
mixtures of n-hexane/acetone (1:1 v/v) or chloroform/methanol
(1:1 v/v), and that the moisture content of the soil should be at
least 5%. The results of a study of the determination of
organochlorine insecticides in three types of soil have been
summarized by Woolson & Kearney (1969). Twelve laboratories
participated using various extraction procedures. In one type of soil
(a silty clay loam) that was fortified with p,p'-DDT at a
concentration of 5 mg/kg, the amounts found in the different
laboratories varied between 1.60 and 5.48 mg/kg. The results of 3 of
the laboratories were discarded by Woolson & Kearney (1969), and the
mean recovery of the other 9 laboratories was 79.3%, with a standard
deviation of 42.2%. Wetting of the soil before extraction was
considered to improve the recoveries, Soxhlet extraction appeared to
be preferable to shaking with the solvent, and hexane/acetone
(1:1 v/v) or hexane/isopropanol (3:1 v/v) appeared better extractants
than other solvents.

A further collaborative study of the determination of organochlo-
rine insecticides in 3 types of soil was reviewed by Woolson (1974).
Although 12 laboratories participated, only 7 completed the study. All
the laboratories used the same extraction, clean-up and quantification
procedures, including premoistening of the soil with ammonium chloride
solution at 0.2 mol/litre. The recoveries of p,p'-DDE, p,p'-TDE,
o,p'-DDT and p,p'-DDT were higher than 80%, with standard errors
of 8-18%. These results are much more consistent than those of the
previous study, and Woolson recommended that premoistening of the soil
with ammonium chloride solution (0.2 mol/litre) followed by extraction
with hexane/acetone (1:1 v/v) should be adopted for the determination
of chlorinated hydrocarbon insecticides in soil.

Several solvents have been used for the extraction of DDT-type
compounds from nonfatty foods such as vegetables, fruit, and cereals;
acetonitrile, alone or mixed with water is used in the method of the
Association of Official Analytical Chemists (AOAC) depending on the
water content of the sample (Horwitz, 1975); Cieleszky et al. (1970)
recommend Soxhlet extraction with diethyl ether.

Maceration of vegetables with a mixture of acetone and hexane was
used by Sissons et al., 1968; the same mixed solvent was used for
cereals, and root vegetables by Abbott et al., 1969 who used
propan-2-ol in the case of fruit and green vegetables. Whiting et al.
(1968), and Skrentny & Dorough (1971) considered a mixture of methanol
and chloroform to be the most efficient extraction solvent for use
with a macerator or a Soxhlet extractor. The acetonitrile extraction
technique was examined by Zerber et al. (1971) for the extraction of
DDT-type compounds from cereal products and feeding stuffs. Diethyl
ether was used by Kucinski (1972) for extraction from canned vegetable
products.

Extraction methods for fat-containing foods are given in the AOAC
Method (Horwitz, 1975), the solvent used, methanol or petroleum ether,
being dependent on the type of sample. Soxhlet extraction using
petroleum ether is recommended by the Federal Health Office of the
Federal Republic of Germany (Anon., 1974); Cieleszky et al. (1970)
also recommended petroleum ether as a suitable solvent.

Smart et al. (1974) compared acetonitrile and dimethyl formamide
as extraction solvents for apples, carrots, potatoes, and vegetables;
a third solvent dimethylsulfoxide was compared with these two solvents
for butter, cheese, and eggs.

Three body tissues or fluids have been analysed in many surveys,
namely adipose tissue, blood (or serum), and mother's milk. The US EPA
Manual (Thompson, 1974) recommends grinding a sample of adipose tissue
with anhydrous sodium sulfate before extraction with petroleum ether;
carbon tetrachloride has been used as a solvent (Mattson et al.,
1953), but it is not appropriate if the final quantification step
involves gas-liquid chromatography and a halogen sensitive detector.
Extraction of DDT-type compounds from blood or serum using hexane has
been described (Dale et al., 1966b), but this extraction procedure is
inefficient, probably as a result of binding by serum proteins. Dale
et al. (1970) investigated a procedure in which the serum was treated
with 97% formic acid before extraction with hexane; experiments using
¹⁴C-DDT indicated that extraction with hexane alone gave results
some 40% lower (based on ¹⁴C activity) than when the serum was first
treated with formic acid. A modified procedure in which whole blood
was treated with 60% sulfuric acid, and then extracted with a
hexane/acetone (9:1) mixture has been reported by Stretz & Starr
(1973). Samples of blood spiked at 4 different levels with p,p'-DDT
were analysed by 11 different laboratories; considerable discrepancies
were found between laboratories and a further study of the method was
considered desirable. Griffith & Blanke (1974) also investigated the
sulfuric acid method, but a microcoulometric detector was used instead
of an electron-capture detector. According to these workers,
consistent recoveries of p,p'-DDT, p,p'-TDE and p,p'-DDE were
obtained in their laboratory but reproducibility between laboratories
was not studied.

The US EPA Manual (Thompson, 1974) method for the extraction of
DDT-type compounds from human milk involves an acetonitrile/hexane
type extraction. A method for extraction from cow's milk, that makes
use of the stability of p,p'-DDT and its derivatives in the presence
of concentrated sulfuric acid, has been published by Coha & Nedic
(1970); the mixture of milk and concentrated sulfuric acid is
extracted with hexane. Prouty & Cromartie (1970) studied the
recoveries of ¹⁴C-DDT in each of the 5 major stages of a method for
determining this compound in the tissues of quail; Soxhlet extraction
with hexane for 6 h, of muscle, liver, heart or brain, after grinding
with sodium sulfate gave recoveries of DDT-type compounds (as ¹⁴C-
activity) of 93-105%. Jonczyk (1970) used hexane to extract DDT-type

compounds from blood and reported recoveries of 67-91%. Acetone was
used as the extracting solvent for DDT-type compounds in the adipose
tissue and brain of partridges (Jonczyk et al., 1970). Wood's method,
using dimethyl sulfoxide, was used by Stec & Juszkiewicz (1972), and
was found to give results for DDT-type compounds that compared
favourably with other methods of analysis of animal tissue, eggs, and
milk.

All methods of extracting DDT also result in the removal of
lipids, if they are present. Regardless of the method of extraction,
the results of most analyses have been reported in terms of fresh or
wet weight of samples no matter whether their lipid content was
extremely low (water and urine), low (soils and most vegetables),
intermediate (milk and many tissues), or high (adipose tissue). In
some instances, samples such as adipose tissue, milk, and, to a lesser
degree, other animal tissues known to contain lipids have been
reported in terms of the concentration of pesticide in extractable
lipid. Because DDT and DDE are known to have a marked affinity for
neutral fat, it was originally supposed that reporting in terms of
lipid would reduce variability within any set of samples by excluding
the influence of connective (and sometimes lymphatic) tissue, which
forms a part of each sample. It appears that variation, as measured by
the coefficient of variation, may not be reduced by this kind of
reporting (Casarett et al., 1968). However, there may be other reasons
for reporting on a lipid basis and it is absolutely essential that the
method of reporting be specified.

2.2.5 Clean-up procedures

The extraction procedures remove not only the DDT-type compounds
from the samples analysed but also coextractives to a greater or
lesser degree according to the type of sample. A number of procedures
have been developed that reduce the amounts of the coextractives
relative to that of DDT-type compounds. If interest is confined solely
to DDT-type compounds, then their stability in the presence of
concentrated sulfuric acid is a very useful clean-up procedure
(Mattson et al., 1953; Czegledi-Janko & Cieleszky, 1968; Murphy,
1972). Usually, however, the concentrations of other compounds are
also of interest and this procedure cannot be used if these compounds
are not stable in concentrated sulfuric acid. Two general clean-up
procedures, used in sequence, are appropriate in these circumstances,
namely, liquid-liquid partition followed by liquid-solid partition.
Liquid-liquid partition systems such as hexane/acetonitrile,
hexane/dimethyl formamide, or hexane/dimethyl sulfoxide are the ones
most commonly used. The liquid-solid partition systems generally
consist of Florisil, silica gel, or alumina as the solid phase, and
hexane or mixtures of hexane and various proportions of a polar
solvent (e.g., diethyl ether) as the mobile phase. Separation of
DDT-type compounds from triglycerides in fat-containing tissues is
achieved with considerable efficiency by liquid-liquid partitions, the

hexane/dimethyl formamide or hexane/dimethyl sulfoxide systems being
generally more efficient than hexane/acetonitrile. Separation of
DDT-type compounds from other organochlorine compounds (e.g., aldrin,
dieldrin, polychlorinated aromatics) or from steroids is not very
efficient using liquid-liquid systems, and the liquid-solid partition
systems should be used in these cases. Separation of DDT-type
compounds from polychlorinated biphenyls is particularly difficult
and, as some of these compounds have similar liquid chromatographic
retention times to those of the various DDT-type compounds, the
analysis of samples containing both classes of compounds requires
considerable care. Detailed clean-up procedures are described by de
Faubert Maunder et al. (1964), Cieleszky et al. (1970), Anon. (1974),
Thompson (1974), and by Horwitz (1975).

The separation of DDT-type compounds from polychlorinated
biphenyls, by liquid-solid (silical gel) partition is discussed by
Armour & Burke (1970), Snyder & Reinert (1971), and Masumoto (1972);
the last-mentioned investigator concluded that a number of factors
required careful control if satisfactory separation of DDT-type
compounds from polychlorinated biphenyls (PCBs) were to be achieved,
in particular, the degree of activation of the silicic acid (irregular
distribution of water molecules onto the silicic acid particles was
also probably important). He found that separation of p,p'-DDE from
four Arochlors was incomplete. A collaborative study of the separation
of DDT-type compounds from PCBs using the Armour & Burke silicic acid
column procedure has been reported (Sawyer, 1973).

Florisil and coconut charcoal have also been investigated for the
separation of PCBs from DDT-related compounds (Reynolds, 1969;
Benvenue & Ogata, 1970; and Stijve & Cardinale, 1974). Another
procedure that has been developed for the separation of DDT-PCB
mixtures is the oxidation of p,p'-DDE to p,p'-dichlorobenzophenone
(Miles, 1972). A method for the determination of p,p'-DDT and a
particular PCB isomer that has similar retention time to that of
p,p'-DDT is based on an empirical relation for p-values (Zelinski et
al., 1973).

2.2.6 Quantitation

2.2.6.1 Determination of DDT-type compounds

Two techniques have played a major role in the quantification of
DDT-type compounds, one is a colorimetric method, the other (now the
most widely used) is gas-liquid chromatography with a halogen-
sensitive detector.

The colorimetric procedure of Schechter-Hailer is described in
detail in the Handbook of the Deutsche Forschungsgemeinschaft (1969),
and by Cieleszky et al. (1970) and Horwitz (1975).

Gas-liquid chromatography is essentially a further-method of
separation of compounds, and, although it is the most effective of the
separation procedures (apart possibly from high pressure liquid-liquid
chromatography) it must be realized that it has its limitations,
particularly if used with a highly sensitive but nonselective detector
such as the electron-capture detector. The basic principles of
gas-liquid chromatography are described by Dal Nogare & Juvet (1962)
and Yermakov (1972), for example, and need not be discussed here.
However, attention is drawn to three aspects of the performance of a
column in the gas-liquid chromatographic separation of DDT-type
compounds from other compounds. First, the DDT-type compounds should
not undergo any thermal degradation or other chemical change; second,
the performance of the column should be assessed by the number of
theoretical plates; and third, the performance of the column as regards
ability to separate p,p'-DDT and related compounds from other
compounds that have similar retention times for a particular liquid
phase must be investigated. Transport of p,p'-DDT and analogues
through a column without chemical change is dependent upon the absence
of reactive centres in the column. This can be attained by using an
inactive solid support (by coating the reactive sites with a silane,
if necessary) and ensuring that the surface of the solid support is
completely covered by an inert liquid phase. The performance of a
column should be checked at regular intervals; the US EPA Manual
(Thompson, 1974) suggests a mixture containing five DDT-type compounds
plus eight other organochlorine insecticides for this purpose. Change
in peak shape (i.e., departure from symmetrical peaks) should always
be regarded as a warning sign. In the case of serious doubt about the
stability of p,p'-DDT or an analogue (which may manifest itself as
a change in the retention time relative to that of aldrin or dieldrin
for example), it is suggested that a fraction collection technique be
used and the identity of the component leaving the column at a
particular retention time with that injected confirmed.

Methods of calculating the number of theoretical plates and of
separation factors are given in standard texts. According to the US
EPA Manual, a column of 2 m length should have about 3000 theoretical
plates. The factors that control the performance of gas-liquid
chromatographic columns are discussed in detail by Scott (1970).
Convenient summaries of the retention times (absolute or relative) of
DDT-type compounds, together with those of other pesticides, using
various column conditions have been published (Yermakov, 1972; Zweig &
Sherma, 1972). Retention times of 51 pesticides relative to that of
aldrin using six stationary phases at three temperatures with an
electron-capture detector have been reported by Thompson et al.
(1975); estimates of the relative retention times at other
temperatures were derived from the relationship between relative
retention time and temperature for each liquid phase. The relative
times of DDT-type compounds and other pesticides on eight stationary
phases were determined by Thompson et al. (1969a). Nonpesticidal

organochlorine compounds that have retention times similar to those of
DDT-related compounds include the polychlorinated biphenyls and
polychloronaphthalenes.

Examples of the close similarity between the relative retention
times of DDT-related compounds and those of various PCB isomers have
been published (Bagley et al., 1970; Richardson et al., 1971; Stijve &
Cardinale, 1974). Goerlitz & Law (1972) demonstrated that there are
also similarities between the relative retention times of DDT-type
compounds and those of various isomers of polychlorinated
naphthalenes. Examples of similarities between the relative retention
times of DDT and its analogues and various components in
polychlorinated biphenyls and polychlorinated naphthalenes was also
reported by Griffith & Blanke (1974). Problems arise in the case of
mixtures of toxaphene and DDT-related compounds (Cahill et al., 1970).

The effects of severe infection loading on column performance,
peak configuration, and conversion of p,p'-DDT into p,p'-TDE, were
studied by Thompson et al. (1969b). These investigators found that the
columns they used could be maintained and restored to full or nearly
full performance capacity by daily changing of the glass injection
port insert and the glass-wool plug at the column inlet.

Two different types of detection systems are most frequently used
for the quantification of p,p'-DDT and analogues after elution from
gas-liquid chromatographic columns, namely, electrochemical detectors
and electron-capture detectors. Two types of electrochemical detector
have been developed, the microcoulometric detector and the micro-
electrolytic conductivity detector, that are considerably more
sensitive to p,p'-DDT and its analogues than the original electro-
chemical detectors. Giuffrida & Ives (1969) described modifications and
improvements in microcoulometric gas chromatography and, in the case
of DDT-type compounds in carrots, they obtained responses from their
microcoulometer that were approximately one-fifth of those given by a
particular electron-capture detector; Griffith & Blanke (1974)
described a microcoulometric method for the determination of
p,p'-DDT, p,p'-TDE and p,p'-DDE in blood. According to Dolan
& Hall (1973), the microelectrolytic conductivity detector can be used
for the selective determination of organochlorine pesticides in the
presence of polychlorinated biphenyls. However, the relative
selectivity in regard to p,p'-DDE does not appear to be as great as
that for other organochlorine insecticides.

The electron-capture detector produces an extremely sensitive
response to organochlorine compounds, but its response is not,
unfortunately, very selective and many other classes of compounds have
electron affinity in the vapour phase. A general review of the
principles and characteristics of the electron-capture detector has
been published by Pellizari (1974). The detector may be used under

direct current or pulse sampling conditions; anomalous responses
obtained in the direct current mode of operation are not present under
pulse sampling conditions (Lovelock, 1963). A major limitation of the
electron-capture detector is that its response is linear over a very
limited range only, but a new mode of operation in which the linearity
extends over about four orders of magnitude of response has been
described by Maggs et al. (1971). For this the detector current is
held constant while the frequency of the applied pulses is varied. The
response of electron-capture detectors is liable to change
significantly during use, and these detectors should be recalibrated
at regular intervals, preferably at least once per day with single
standard injections at frequent intervals between injections of
extracts from the samples under investigation. It is of interest that
Mendoza (1971) reported a significant difference in the response of a
gas-liquid chromatograph to p,p'-DDT when injections were made at
fast and slow rates.

The combination of mass-spectrometry with gas-liquid chromato-
graphy was mentioned in section 2.2.4 as a means of confirming the
identity of residues of DDT-type compounds. This combination of
instruments can also be used to quantify the amounts present. The
total ion current corresponding to mass fragments of appropriate mass
number (m/e; in the case of p,p'-DDE, 316, 318, 246 and 248) is
measured and the amount present is calculated from the calibration of
the instrument, preferably by an internal standard. Palmer & Kolmodin-
Hedman (1972) determined the concentration of p,p'-DDE in human
plasma by mass-fragmentography using the ion current corresponding to
m/e 216 and 218; the results showed an excellent agreement with the
values obtained using an electron-capture detector.

Semiquantitative methods of the determination of p,p'-DDT and
its analogues include paper chromatography and thin-layer chromato-
graphy; these methods, which are more appropriately used for the
confirmation of identity of residues, are described by McKinley (1963)
and Sherma (1973) respectively. Bishara et al. (1972a) have given the
tlc R_f values for p,p'-DDT and 14 related compounds using 33 solvent
systems.

2.2.6.2 Determination of p,p'-DDA in urine

A major metabolite of p,p'-DDT, excreted in the urine, is bis-
(4-chlorophenyl)acetic acid ( p,p'-DDA). A method has been developed
by Cranmer et al. (1969), based upon the extraction of p,p'-DDA from
acidified urine, conversion into the methyl ester, Florisil column
clean-up, and gas-liquid chromatographic analysis of the ester. A
detailed outline of this procedure is given by Thompson (1974). The
sensitivity of response of the methyl ester is not high, the limit of
detection being about 2 ng. Cranmer & Copeland (1973) used the

2-chloroethanol ester, the response of an electron-capture detector
being about 3.7 times greater than that of the methylester (i.e.,
limit of detection about 0.5 ng). An advantage of this ester is that
it is well separated from p,p'-DDT, whereas the methyl ester has a
retention time similar to that of p,p'-DDE. The retention time of
the pentafluorobenzyl ester compound is about twice that of p,p'-DDT
and the response of an electron-capture detector is considerably
greater than that for the 2-chloroethanol derivative (Johnson, 1973).

2.2.6.3 Method of reporting results

Francis Galton (1879) pointed out that many vital effects are
distributed logarithmically; his paper was followed by a technical one
(McAlister, 1879) presenting the mathematics. Apparently Robinson &
Hunter (1966) were first to point out that this principle applies to
the storage of pesticides so that the geometric mean is a more
appropriate parameter than the arithmetic mean for expressing
insecticide content. In recent years, increasing use has been made of
the geometric mean as recorded by footnotes in Table 9. Few of the
arithmetic means that have been reported are so much in error that
they should be discarded. As Galton (1879) noted, the difference
between the arithmetic and geometric mean is small if the range of the
values averaged is narrow.

2.2.7 Validation of analytical methods for DDT-type compounds

Ideally, an analytical method should be accurate and highly
precise. A study of the accuracy and precision of art analytical
method must be made in order to assess the relationship between the
actual amount present and the results obtained in practice; such a
study may be called the validation of the procedure. There are two
major types of validation procedure. The first type requires the use
of radio-labelled molecules of p,p'-DDT, e.g., ¹⁴C p,p'-DDT.
Prouty & Cromartie (1970) determined the ¹⁴C-activity in muscle,
liver, heart, and brain of quail that had been given ¹⁴C-labelled
p,p'-DDT. Four of the steps in the analytical procedure were studied
and it was concluded that the major discrepancies occurred during the
elution and concentration steps from Florisil columns and from zones
of thin-layer chromatography plates. Chiba & Morley (1968) used ¹⁴C-
labelled p,p'-DDT in a study of soil analysis.

The other validation procedure, which has been studied more
intensively, involves the comparison of the results of analyses with
the values for samples fortified with accurately known amounts of
p,p'-DDT or analogues. There are several variations of this
validation procedure, all comparing the fortified (nominal) value with
the results of different methods in the same laboratory, with the
results of a specified method carried out in different laboratories,
or with the results of different methods in different laboratories.

Versino et al. (1971) compared 8 clean-up procedures for test
mixtures containing 10 pesticides. Two extracts, from apples and
lettuce, were fortified (spiked) with p,p'-DDT at 0.2 mg/litre,
p,p'-TDE at 0.1 mg/litre, and o,p'-DDT at 0.1 mg/litre plus
dieldrin and 7 organophosphate insecticides. The recoveries for the 8
column clean-up procedures ranged from 89-100% for p,p'-DDT, 85-101%
for p,p'-TDE, and 95-100% for o,p'-DDT. It should be noted that
this investigation did not include a study of extraction efficiency
and that the extracts contained only small amounts of lipids. In
studies by Smart et al. (1974), samples were analysed of milk, butter,
cheese, eggs, apples, potatoes, carrots, and cabbages, fortified with
p,p'-DDT, p,p'-TDE, and p,p'-DDE (and 5 other organochlorine
insecticides) at concentrations corresponding to those suggested as
limits by the Codex Alimentarius Commission. Five replicate analyses
of each sample were made, using up to 4 procedures, the final step of
each analytical procedure being gas-liquid chromatography/electron-
capture detection, with 3 different stationary phases. These workers
concluded that there were no gross discrepancies in their results.
However, they did not use the concept of "total error" (see above) in
the discussion of their results, and the total errors for the
determination of the 3 DDT-type compounds, calculated from their
results, were in the range of 12% to 95%.

Results have been reported (Carr, 1970) of a collaborative study
by 10 laboratories of the AOAC method for the determination of 4
DDT-type compounds in samples of butterfat fortified by the addition
of these 4 compounds at 2 different concentrations. The mean recoveries
varied from 86-108%, and the coefficients of variation between
laboratories were in the range 7-28%. The total errors for the 2
levels of fortification respectively were: p,p'-DDT, 22 and 14%;
p,p'-TDE, 17 and 30%; p,p'-DDE, 17 and 36%; and o,p'-DDT, 30 and
43%.

Carr (1971) gives the results of a collaborative study by 8
laboratories of the analysis of fish samples fortified (2
concentrations) with p,p'-DDT, p,p'-TDE and p,p'-DDE, and 3 other
organochlorine insecticides. On the basis of a ranking technique
(Youden & Steiner, 1975), the results of 2 laboratories (Nos. 2 and 8)
for the 6 compounds were rejected as outliers. However, if the results
for the 3 DDT-type compounds are considered, then laboratories 7 and 8
gave results that are outliers. The total errors at the 2
concentrations for the 6 laboratories not rejected by Carr were:
p,p'-DDT, 15 and 36%; p,p'-TDE, 32 and 32%; p,p'-DDE, 26 and 39%.

Sawyer (1973) reported a collaborative study by 9 laboratories of
samples of red salmon fortified with 3 DDT-type compounds and PCBs.
The analyses were carried out without and with silicic acid column
separation of PCBs from DDT-type compounds. The total errors without

and with silicic acid separation, were 34% and 43% respectively for
p,p'-DDT, 50 and 47% for p,p'-TDE and 35 and 36% for p,p'-DDE.
Very similar results were obtained with another type of PCB/DDT
mixture.

Samples of soil, fortified with 4 DDT-type compounds plus 6 other
organochlorine insecticides were analysed in 7 laboratories (Woolson,
1974); the total errors (all collaborators) were: p,p'-DDT, 17-33%;
p,p'-TDE, 19-37%, p,p'-DDE, 18-42%; o,p'-DDT, 28-56%.

Fifteen laboratories collaborated in a study of the determination
of p,p'-DDE in eggs, and p,p'-DDT, p,p'-DDE and o,p'-DDT in
kale (Finsterwalder, 1976). Five laboratories analysed eggs by the
AOAC method, and the total error was 21.1%; the total error for 10
laboratories using a modification of the AOAC method was 30.4%. The
results of one laboratory for analyses of kale were rejected as
outliers, and the total errors for the other 14 laboratories were:
p,p'-DDT, 19.7%; p,p'-DDE, 18.8%; o,p'-DDT, 17.4%.

An international collaborative programme on methods of analysis of
organochlorine insecticides has been sponsored by the Organization for
Economic Cooperation and Development (OECD), and the results of 2
studies have been published. In the first (Holden, 1970), a solution
in hexane of 6 organochlorine compounds, 3 being DDT-related
compounds, was analysed by 17 laboratories in 11 countries. The total
errors for p,p'-DDT, p,p'-TDE and p,p'-DDE were in the range
14-21%, the major component in this total error being the standard
deviation of results from different laboratories, probably indicating
errors of calibration. In the second study (Holden, 1973), a sample of
corn oil fortified with 4 DDT-type compounds and 3 other
organochlorine insecticides was analysed in 19 laboratories in 10
countries: the mean total error for the 4 DDT-type compounds (after
excluding 3 outliers) was 35%.

Some of the results of this collaborative validation study are
very satisfactory, but many of them, as judged by the criterion of
total error, are unsatisfactory, even when outliers are rejected, and
they illustrate the need for scrupulous care by analysts: the use of
clean glassware, chemicals of established purity, and constant
checking of the performance of liquid-solid partition columns and of
instruments.

2.2.8 Analytical methods for the evaluation of the biochemical effects
of p,p'-DDT and its analogues

The increased activity of enzymes, particularly in the hepatic
endoplasmic reticulum, if the exposure to p,p'-DDT and its analogues
is sufficiently great, has been recognized in recent years.
Measurement of such changes in enzyme activity may be made by studying
changes in the rates of metabolism of certain drugs (such as

antipyrine), but methods that do not require the administration of a
drug (and the collection of blood samples if the plasma half-life is
used as the biochemical parameter) are preferable. Two methods, in
which the concentration of either 6-ß-hydroxycortisol or D-glucaric
acid in the urine is measured have been developed. A procedure for the
determination of 6-ß-hydroxycortisol in urine has been described by
Kuntzman et al. (1968); and one for D-glucaric acid by Marsh (1963)
(see section 8.2.5.1).

These enzyme induction changes are not specific for DDT-type
compounds.

3. SOURCES OF ENVIRONMENTAL POLLUTION

3.1 Discovery and Introduction

DDT does not occur naturally. It was first synthesized by Zeidler
as reported in 1874. However, it was not put to any use until its
insecticidal properties were discovered by Paul Müller in 1939. The
Swiss patent was issued in 1942.

As an example of the speed with which DDT was developed and used,
the first sample sent to the USA arrived there in September 1942. This
sample was tested for effectiveness and safety. The results were so
encouraging that manufacture was given high priority. At first, the
entire production was used for the protection of troops against
malaria, typhus, certain other vector-borne diseases, or against
biting flies or other insects that are merely pests. As the supply
increased, DDT was used in the USA for control of malaria in military
areas, that is in the vicinity of military installations, ports, and
transportation centres. As a result of this effort, mosquito
transmission of malaria was brought to an end in the USA in 1953, even
though military personnel and other infected persons from the tropics
continued to reintroduce the disease extensively as late as 1972 and
to a lesser degree thereafter.

3.2 Production and use

The revolution in the control of malaria and typhus among allied
troops and among certain civilian populations during World War II was
accomplished with relatively little DDT. Far greater amounts were
required for the control of agricultural and forest pests and this
became possible when the compound was released in the USA for
commercial use on 31 August 1945. Civilian use in other countries
became possible a little later, first, largely on the basis of
importation and gradually on the basis of local manufacture.
Unfortunately, there is apparently no record of world production of
DDT. Production and use in the USA is shown in Table 2.

Quantities of DDT and related compounds used in or sold for
agricultural purposes in 1970 were as follows (metric tonnes):
Australia (about 1000); Austria (20.5); Botswana (2.0); Canada
(287.0); Columbia (980.0); Czechoslovakia (270.0); Democratic
Kampuchea (46.8); Egypt (3457.0); El Salvador (466.0); Federal
Republic of Germany (152.0); Finland (6.1); Ghana (0.3); Guatemala
(380.0); Hungary (20.6); Iceland (0.3); Israel (10.0); Italy (2178.0);
Japan (401.0); Kuwait (0.2); Madagascar (176.0); Ryukyu Islands
(Japan) (0.3); Sri Lanka (16.6); Sudan (269.0); Upper Volta (1.5); and
Uruguay (5.0) (FAO, 1972). These values total 10 146.2 metric tonnes.
Thus, at least until very recently, the use of DDT was extensive on a
worldwide basis but varied greatly from one country to another.

Table 2. Metric tonnes of DDT produced and used in the USA^a

Year Production Use

1944 4 366
1945 15 079
1946 20 220
1947 21 534
1948 9 181
1949 19 822
1950 35 448
1951 48 144
1952 45 327
1953 38 268 28 349
1954 44 088 20 465
1955 56 760 28 032
1956 62 441 34 194
1957 56 136 32 205
1958 65 920 30 255
1959 71 097 35 771
1960 74 471 31 818
1961 77 763 29 061
1962 75 764 30 502
1963 81 154 27 744
1964 56 113 22 925
1965 63 859 24 034
1966 64 115 20 685
1967 46 906 18 260
1968 63 231 14 848
1969 55 839 13 724
1970 26 860 11 316

^a Based on data from US Tariff Commission (Hayes, 1975).

3.3 Changing Patterns of Use

Before 1945, all of the DDT produced in the USA for example, was
used or allocated by the military services for various medical and
public health uses. Early in 1945, it became available tor rather
extensive experimental work in agriculture, and it was commercially
available in limited quantities early in the autumn of the same year
(US Department of Agriculture, 1945a,b). The results were so
spectacular that use increased until 1959. In response to a demand for
exports, production continued to increase to about 1963. Even before
then some restrictions were placed on its use, mainly to minimize
residues in food and in the feed of animals that produce milk and

meat. Among the first of these restrictions was that on its use on
dairy cattle or in dairy barns (USDA, 1949). Another important factor
reducing the use of DDT was the increasing resistance of pests. One of
the first species to be affected was the house-fly; because of its
abundance and widespread distribution, its resistance was bound to be
noticed by the public, generally. Although many pests of public health
importance became resistant to DDT in some or all of their range,
resistance among vectors of malaria was less marked. Because malaria
control constitutes such a large segment of vector control, the use of
DDT for vector control has tended to remain stable, while its use in
agriculture has continued to decline, especially in temperate
climates.

The ban on the use of DDT and certain other organochlorine
insecticides in Sweden from 1 January 1970 was based on a number of
ecological considerations.

Government agencies of some other countries attempted to justify
severe restrictions on the use of DDT by alleging that it was a threat
to human health. This was in response to ecologists who considered
that the widespread occurrence of DDT in the environment was
inherently bad and was the direct cause of injury to certain fish and
birds. However, this did not prevent the same agencies from making a
proviso that DDT might be used, if needed, to combat any future threat
from vector-borne disease within their boundaries.

Of course, before the restrictions were put into effect, it had
already been possible to eradicate malaria in the USA and Italy, for
example, and to control for the first time an epidemic of typhus in
Italy and Germany (Simmons, 1959). Apparently, there have never been
accurate figures on the proportion of the compound used for
agriculture and for public health even in those countries that have
recorded their total use of the compound.

As the situation now stands, DDT is still used extensively, both
in agriculture and for vector control, in some tropical countries.
Apparently, information is not available on how much of the
agricultural use involves food protection or how much loss of food
production would result if the use of DDT were discontinued. The
picture with malaria control is clear. Substitution of malathion or
propoxur for DDT would increase the cost of malaria control by
approximately 3.4- and 8.5-fold, respectively, and this increase could
not be supported. If DDT were not used, vast populations in the
malarious areas of the world would be condemned to the frightening
ravages of endemic and epidemic malaria (WHO, 1971).

4. ENVIRONMENTAL TRANSPORT AND DISTRIBUTION

Because DDT has been sprayed on people, domestic animals,
buildings, agricultural crops, and forests, it is not surprising that
it is now distributed widely in the environment. During the
application of any spray or dust to a field, it is often possible to
observe drift of the particulate material. This is especially true if
the application is made by aircraft or by ground equipment that shoots
the spray to the top of orchard trees. If the application is made to
forests, it is very likely that at least part of the spray will fall
directly on streams or lakes. Following application, redistribution is
inevitable. As discussed more fully in the following sections, some
DDT in soil enters the air by evaporation or on wind-blown dust. Even
if watercourses are avoided initially, some insecticide will be washed
into them by rains, mainly in conjunction with soil particles.

4.1 Local Drift in Air

The fact that there is drip is implicit from measurements of DDT
on surfaces just after application. Wilson et al. (1946b) recovered
only 7.5% of the nominal dose from plants; this proportion is typical
but ignores material that may have fallen between plants or between
leaves and come to rest on the ground. Of greater interest are
measurements of total recovery made by means of absorbent targets laid
out in advance. As might be expected, recovery of aerosols is less
than recovery of sprays when other conditions of application are
similar. Only 12.5% was recovered at the centre of swaths following
application of a thermal aerosol by aircraft and recovery decreased at
increasing distances from the centre (Hess & Keener, 1947). In another
study, recovery was 8% for a thermal aerosol and 46% for a spray
(Scudder & Tarzwell, 1950). In a different situation, the average
seasonal recovery for DDT applied as a thermal aerosol ranged from 10
to 12%, while that for spray ranged from 56 to 76% (Tarzwell, 1950).
However, somewhat lower recoveries of DDT sprays have frequently been
reported: 30% (Hoffman & Merkel, 1948), 39% (Hoffman & Surber, 1948),
and 27% (Surber & Friddle, 1949).

Most DDT particles that miss the target for which they are
intended would be expected to fall in the general area. Studies of
residues in soil and in wildlife indicate that this is true. The
logarithm of the concentration of DDT-related compounds in soil
samples collected in a desert area downwind from an area of intensive
agriculture showed an almost straight line inverse relationship to the
logarithm of the distance from the source. The soil levels were about
1 mg/kg at 10 m and about 0.001 mg/kg at 100 000 m from the point of
application. The concentrations of DDT in the tissues of wildlife were
proportional to its concentrations in the soils of their habitats
(Laubscher et al., 1971). Where cultivated fields to which DDT has
been applied for years are interspersed among pastures and other
fields where DDT is not used, the uncultivated soils contain only a

little less DDT than the cultivated ones. In one study, it was found
that most of the DDT was in the top 2.5 cm, but samples for comparison
were taken to a depth of 7.5 cm; the average concentrations