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

    Participantsa

     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 DDTa 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]
    DDEa                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]
    DDMUa               1,1'-(2-chloroethenyldene)-             -Cl       None      =CHCl
                        bis[4-chlorobenzene]-
    DDMSa               1,1'-(2-chtoroethylidene)-              -Cl       -H        -CH2Cl
                        bis[4-chlorobenzene]
    DDNUa               1,1'-bis(4-chlorophenyl)ethylene        -Cl       None      =CH2
    DDOHa               2,2-bis(4-chlorophenyl)ethanol          -Cl       -H        -CH2OH
    DDAa                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

    chlorobenzilatec    ethyl 4-chloro-alpha-(4-chlorophenyl)-  -Cl       -OH       -C(O)OC2H5
                        alpha-hydroxybenzeneacetate

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

    methoxychlorc       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
    Rf 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 Rf 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 USAa

    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 were 0.75
    to 2.03 mg/kg in cultivated soil and 0.10 to 0.91 mg/kg in
    uncultivated sod (US Department of Agriculture, 1966). These
    concentrations may be compared (by mathematical calculation alone) to
    1.0 mg/kg, the concentration resulting from mixing DDT into soil
    uniformly and with no loss whatever following application at the rate
    of 1.12 kg/ha, the standard specific gravity (1.47) of soil and a
    depth of 7.6 cm being assumed. The range of specific gravities for
    most agricultural soils is 0.4 to 2.0 and the corresponding depths
    yielding 1 mg/kg are 28 to 5.60 cm. It is of interest that the
    residues in cultivated soft were of the order of magnitude that would
    be expected from a single application, even though the average
    cumulative rate of application during the last 10 years (11.2 kg/ha)
    had been 10 times as great.

    4.2  Distant Drift in Air

        Dust bearing DDT at a concentration of 0.6 mg/kg has been observed
    about 1600 km from its area of origin. Other pesticides were also
    present in this dust which had settled out on a recently rain-rinsed
    roof. The cloud of dust was so dense and unusual that its progress was
    reported in newspapers, as the storm that mobilized it in Texas
    carried it at least as far as Ohio, where the sample was collected
    (Cohen & Pinkerton, 1966).

        Dust collected on the island of Barbados by means of nylon nets
    treated with 50% aqueous glycerine was assumed to have been blown from
    Africa, a distance of over 4850 km. Samples of the dust contained
     p,p'-DDT most frequently and in greatest concentration, but the
    concentration of all pesticides was only 0.001 to 0.164 mg/kg with an
    average of 0.041 mg/kg (Risebrough et al., 1968).

        DDT evaporates from sprays and dusts at the time of application
    and at any time that dust bearing the compound is mobilized by the
    wind. Furthermore, DDT can be detected over treated fields for more
    than six months after application, and there is a concentration
    gradient from the soil upward (Willis et al., 1971). Under these
    circumstances, it is obvious that DDT is transported in the form of a
    vapour as well as by means of dust. However, if samples are collected
    where no application of DDT has been made, identification of the
    origin of the vapour or measurement of the distance it may have
    travelled is even more difficult than with dust.

        Evidence that DDT in one form or another has travelled great
    distances and is, in fact, worldwide in its distribution has been
    deduced from finding it in the rainwater of remote, nonagricultural
    places (Tarrant & Tatton, 1968) or in water melted from Antarctic snow
    (0.00004 mg/litre) (Peterie, 1969). In such remote places as

    Eskdalemuir in Scotland and Lerwick in the Shetland Islands, the
    average concentrations of  p,p'-DDT in rainwater (0.000030 and
    0.000046 mg/litre, respectively) were not greatly different from the
    averages (0.000018-0.000066 mg/litre) found in widely separated
    agricultural areas, suggesting that the compound is rather evenly
    distributed in the air (Tarrant & Tatton, 1968).

    4.3  Distribution in Water

        DDT has a strong tendency to adsorb on surfaces. Most DDT that
    enters water is already firmly attached to soil particles, and remains
    attached. It was shown very early that, if DDT does find its way into
    clear water, it is gradually lost by adsorption on surfaces (Carollo,
    1945). The sediments in water tend to move downstream and eventually
    to enter estuaries. Of the various chlorinated hydrocarbon
    insecticides, DDT and its metabolites are the ones most commonly
    found, but the residues tend to be low (Butler, 1969). In fact, in an
    estuary associated with the Mississippi River, the levels of
    pesticides decreased strikingly from the early 1960s to the late 1960s
    (Rowe et al., 1971).

    4.4  Bioaccumulation of DDT and Its Degradation in the Environment

        Many studies of DDT and related compounds in the environment have
    focused on organisms and locations in which concentrations of DDT have
    been observed to increase. Concentration in living organisms may be
    the result of adsorption from water, of the filtering out of algae or
    detritus bearing the compound, or of biological magnification in the
    strict sense, that is progressive accumulation in different steps of a
    food chain. Although the mechanisms are poorly understood, observation
    has shown that residues do reach an equilibrium and sometimes decline.
    For example, where DDT was applied to cotton at a cumulative rate of
    11.2 kg/ha so that a residue of over 10 mg/kg in the soil would be
    expected after many years of continual use, the actual residues ranged
    from 0.75 to 2.03 mg/kg (USDA, 1966). An example of decreased residues
    was seen in the grebes of Clear Lake (Rudd & Herman, 1972).

        Evidence is accumulating that the disintegration of DDT may be
    rapid in some situations. Under biologically active, anaerobic
    conditions as little as 1% of DDT remained after 12 weeks of
    incubation (Hill & McCarty, 1967; Guenzi & Beard, 1968). Probably more
    important is the disintegration of DDT under the influence of
    ultraviolet light. Hartley (1969) pointed out that much of any
    pesticide vapour escaping to 50 metres or more above the ground will
    ascend even higher by eddy diffusion and eventually reach the
    photochemically active ionosphere. The rapid destruction of DDT by
    ultraviolet light under laboratory conditions has been demonstrated

    (Mosier et al., 1969; Plimmer et al., 1970; Miller & Narang, 1970;
    Plimmer & Klingebiel, 1973; Crosby & Moilanen, 1977). Gab et al.
    (1975, 1977) offered evidence that DDT and DDE are converted to carbon
    dioxide and hydrochloric acid; the destruction was so complete that
    they characterized it as photomineralization. In spite of very real
    progress in understanding the fate of DDT in the environment (see
    Annex), much more work will be required before a quantitative balance
    can be measured between addition of the compound and its
    disintegration.

    5.  ENVIRONMENTAL EXPOSURE LEVELS

    5.1.  Exposure of the General Population

    5.1.1  DDT in air

        In spite of the generalization in section 4.2 that DDT is rather
    evenly mixed in the air, some increase in concentration may be noted
    in connexion with the time and place of application. The highest
    concentration of insecticide found in the air of communities with
    anti-mosquito fogging programmes was 0.0085 mg/m3 (Tabor, 1966).
    Concentrations one or two orders of magnitude greater have been
    reported for several insecticides in the breathing zone of orchard
    spraymen, and values of 1.2-0.26 mg/m3 have been found at distances
    of 500-5000 m from ground spraying (Belonozko et al., 1967). In six
    small communities in an agricultural area in the USA, DDT was found in
    concentrations ranging from 1 × 10-6 to 22 × 10-6 mg/m3 (Tabor,
    1966). Substantially higher values (0.002-0.05 mg/m3) were reported
    for centres of population in the USSR (Belonozko & Kucak, 1974). In an
    urban location in a generally nonagricultural area of the USA, the
    highest concentration found was 2.36 × 10-6 mg/m3 (Antommaria et
    al., 1965). The combined concentrations of DDT, dieldrin, and lindane
    in the Munich area of the Federal Republic of Germany in 1971 were
    even lower, only exceptionally rising as high as 1 × 10-6 mg/m3
    (Weil et al., 1973).

        With few exceptions, the highest average concentration of DDT in
    air to which workers are exposed regularly (7.1 mg/m3) is that
    associated with spraying the interior of houses (Wolfe et al., 1959).
    However, concentrations ranging from 2 to 104 mg/m3 have been
    reported in places where DDT dust was prepared and packed (Medved' et
    al., 1975).

    5.1.2  DDT in water

        Under agricultural conditions, the concentration of DDT in water
    may be high. For example, 0.01 mg/litre was found in the runoff from
    melting snow from fields where sugar-beets had been grown (Medved' et
    al., 1975).

        The highest level at which DDT has been found in rainwater in an
    urban area during a period of a month is 0.0004 mg/litre (Abbott et
    al., 1965). The highest concentration reported in potable water
    (0.02 mg/litre) occurred some years ago (Middleton & Lichtenberg,
    1960). In a much more comprehensive study made a few years later, the
    highest concentration of any insecticide was found (0.00012 mg/litre),
    but this was dieldrin and not DDT (Weaver et al., 1965). Many samples
    did not contain detectable insecticide of any kind. A study of surface
    waters in the USA during the years 1964-1968 indicated that the
    residues reached a peak in 1966 and then dropped sharply in 1967 and

    1968, in spite of improved analytical methods. The highest value for a
    DDT-related compound in those years was 0.00084 mg/litre (Lichtenberg
    et al., 1970). By 1971, the concentration in the Federal Republic of
    Germany was even lower, averaging 0.00001 mg/litre and never going as
    high as 0.001 mg/litre (Weil et al., 1973). The average values for
    total DDT in drinking water in Czechoslovakia were 0.000011 and
    0.000015 mg/litre in 1972 and 1973, respectively (Hruska & Kociánová,
    1975). DDT was not detected (<0.0000000166 mg/litre) in tap water in
    a recent survey carried out in Ottawa, Canada (McNeil et al., 1977).

    5.1.3  DDT in food

        Residues of DDT were measured as early as 1945 (before the
    compound was available commercially) on apples to which it had been
    applied experimentally for the control of the coddling moth (Harman,
    1946). Apparently, the earliest effort to learn how much DDT the
    average man obtains from his daily food was that of Walker et al.
    (1954), who reported that the amount of insecticide in restaurant
    meals in Wenatchee, Washington, USA indicated an average intake of
    0.184 and 0.102 mg/man per day for DDT and DDE, respectively. Soon,
    other studies revealed similar levels of DDT intake for persons who
    ate an ordinary range of foods but who lived in different parts of USA
    (Hayes et al., 1956; Durham et al., 1965b). Most of the DDT was in
    food of animal origin, and persons who abstained from eating meat but
    obtained the food they ate from regular, commercial sources received
    an average of only 0.041 and 0.027 mg/man per day of DDT and DDE,
    respectively (Haynes et al., 1958). The difference did not depend,
    however, on meat  per se, for no DDT and only traces of DDE were
    found in the meat and other products obtained from Arctic wildlife
    that constituted much of the diet of Eskimos (Durham et al., 1961).

        Following restrictions on the application of DDT to livestock, to
    their barns, and to the forage crops on which they fed, there was a
    gradual decrease in residues in animal products used as human foods.
    Restrictions on the use of DDT on crops eaten directly by man resulted
    in reduced residues in vegetable foods. Complete meals collected
    mainly from the same restaurants in Wenatchee and analysed in the same
    laboratory indicated that, by 1964, DDT intake was only 0.038 mg/man
    per day (Durham et al., 1965b) compared with 0.184 mg/man per day
    reported by Walker et al., 11 years earlier. Thus, intake had been
    reduced to less than one-fourth. A further reduction to about one-
    eighth of the 1953-54 values was indicated by the nationwide study by
    the US Food and Drug Administration, usually called the Market Basket
    Survey. Intakes of DDT indicated by this study for the succeeding
    years 1965-70, were 0.031, 0.041, 0.026, 0.019, 0.016, and
    0.015 mg/man per day, respectively (Duggan & Weatherwax, 1967; Duggan,
    1968; Duggan & Lipscomb, 1969; Duggan & Corneliusen, 1972).

        The widespread shipment of food in the USA tends to explain the
    fact that food residues are generally similar in different parts of
    the country. However, small differences do exist and may be accounted

    for by the fact that, on average, meat is not shipped as far as
    vegetables. Much of the feed for livestock is produced on the same
    farm or at least in the same area in which the animals are raised.
    Whatever the reason, the coefficients of correlation between latitude
    and human intake were -0.63 and -0.59 for the sampling periods 1966-67
    and 1967-68, respectively (Hayes, 1975).

        Early studies (Swackhamer, 1965) indicated that both the frequency
    and concentration of residues were slightly less in Canada than in the
    USA. More recent studies in Canada similar to the Market Basket Survey
    indicated total DDT dietary intakes of 0.018, 0.011, 0.011, 0.007 and
    0.007 mg/man per day for the years 1969 to 1973, respectively (Smith,
    1971; Smith et al., 1972, 1973, 1975).

        The analysis of whole meals collected in south-east England during
    1965 and 1966 gave results similar to those obtained during the same
    period in the USA; the calculated daily intakes in England were 0.030
    and 0.025 mg/man per day for DDT and DDE, respectively (McGill &
    Robinson, 1968).

        There may be considerable differences in intake in different parts
    of the same country. Hruska & Kociánová (1975) reported that the
    average intake of DDT plus DDE in 1972 was 0.002 mg/man per day in
    southern Bohemia and was 0.099 mg/man per day in Slovakia. Striking
    differences may exist between urban and rural areas (Almeida et al.,
    1975).

        Values for total DDT-related compounds in regular food in the USSR
    are not available, but the separate values for DDT and DDE in daily
    diets from different regions shown in Table 3 suggest that the total
    may be higher than in the UK and USA and that the amount of DDE may
    exceed the amount of DDT. Both high total values and an unusually high
    proportion of DDE would be consistent with extremely high total values
    a few years earlier and recent discontinuation of the use of DDT. In
    fact, in the Soviet Union, DDT has been eliminated from the list of
    pesticides recommended for use in agriculture since 1970. During the
    period 1966-69, 0.8% of food samples contained residues as high as
    5.1 mg/kg, and 4.2% contained residues over 1.0 mg/kg.

        Although total intake of DDT from food has not been measured in
    some parts of the world, worldwide measurements of storage of DDT and
    its metabolites in human body fat indicate that the extremes of total
    exposure have varied by a factor of about 10, but that total exposure
    for most populations has varied by a factor of no more than 3 (see
    Table 9).

    Table 3.  Residues of DDT and DDE in daily cooked diets from
              different areas of the USSR during 1971/72a

    Area           No. of       Range of residues of
                   daily                                           
                   diets        DDT                DDE
                   examined     (mg/diet)b         (mg/diet)b

    North          121          0.00006-0.0103     0.0001-0.0093
    West           191          traces-0.094       traces-0.039
    South-east      62          traces-0.15        traces-0.330
    South I        184          0.02-0.260         0.020-0.5
    South II        51          traces-0.052       traces-0.132

    a  From: Medved' et al. (1975).
    b  Equivalent to mg/man per day.

        It is important to note that local practice may result in high
    residues in the food of one or more families even though residues are
    low in commercially produced food available in the same area. Thus
    Durham et al. (1965b) reported that the average DDT intake from
    household meals in Wenatchee was 0.314 mg/man per day at the same time
    that restaurant meals in that town contributed only 0.038 mg/man per
    day. The difference was largely the result of very high residues in
    some eggs eaten by local families; the chickens foraged near orchards,
    which had been treated with DDT. The restaurants used commercially
    available eggs and not those produced locally. In a similar way,
    practices peculiar to one country may account for high residues in
    some of their food. For example, very high residues of DDT were
    reported in some samples of staple foods in India (Sharangapani &
    Pingale, 1954). Dale et al. (1965) suggested that the high levels of
    DDT that they found in some Indians might be the result of direct
    addition of the compound to staple food to prevent insect infestation,
    even though the practice did not have government approval.

    5.1.4  Miscellaneous sources

        It has been suggested that there is a positive correlation between
    the use of household insecticides and the concentration of DDT in
    house dust, on the one hand, and the storage of DDT in people on the
    other (Deichmann & Radomski, 1968; Radomski et al., 1968; Davies et
    al., 1969b, 1975; Edmundson et al., 1970b). However, another study of
    dust in 16 urban households, 4 farm households, and 8 households in
    which at least one member was a pesticide formulator, failed to reveal
    a statistically significant correlation between the levels of various
    pesticides in dust and in the serum of people living in the homes.

    There were striking individual examples of workers whose homes
    contained high concentrations of the compounds they used
    professionally and other examples in which there was circumstantial
    evidence relating household dust residues to body burden (Starr et
    al., 1974).

        There can be no doubt that insecticides used in the household or
    introduced on the clothing of workers are important sources of intake
    of DDT in some instances. It is not clear whether the relevant
    absorption involves mainly the inhalation of dust, the contamination
    of food within the home, or even dermal absorption.

    5.1.5  Relative importance of different sources

        It has been estimated (Campbell et al., 1965) that over 90% of the
    DDT stored in the general population is derived from food. About 1965,
    intake in the USA was approximately 0.04 mg/man per day from food,
    less than 0.000046 mg/man per day from water, less than 0.00006 mg/man
    per day from urban air and less than 0.0005 mg/man per day from air in
    small agricultural communities. The reason for the qualification "less
    than" is that the intakes were calculated from the highest
    concentrations reported in drinking-water and air because no average
    values were established.

        Other investigators (Durham et al., 1965b; Morgan & Roan, 1970;
    Medved' et al., 1975) concluded independently that ordinary food is
    the major source of DDT and related compounds for the general
    population. Intake from ordinary food is a base to which other kinds
    of intake -- including that from exceptional food -- may be added. An
    example of such an addition -- eating eggs from chickens allowed to
    run loose in DDT-treated orchards has already been discussed in
    section 5.1.3.

    5.2  Exposure of Infants and Young Children

        Babies tend to be born with slightly lower blood levels of DDT
    than are found in their mothers (O'Leary et al., 1970b; Schvartsman et
    al., 1974, see also section 6.2.2). This simply indicates that the
    placenta excludes some but not all of the DDT available to it. During
    the first 10 or 15 years of life, DDT storage levels rise to the adult
    population level (Hayes et al., 1958; Hunter et al., 1963; Wassermann
    et al., 1965, 1967; Robinson et al., 1965; Davies et al., 1968, 1969a;
    Watson et al., 1970).

        It has been known for a long time that human milk may contain a
    higher concentration of DDT than cow's milk in the same country (Egan
    et al., 1965; Quinby et al., 1965b; Ritcey et al., 1972; Olszyna-
    Marzys et al., 1973). So far, there is no evidence that this small
    difference is of any significance for breast-fed compared with bottle-
    fed babies. This is even true in places where the concentration of DDT
    in human milk is comparatively high (see section 6.3.1.4).

        On the average, the incidence and levels of residues in
    commercially prepared food for babies in the USA are lower than those
    in raw agricultural products, other processed foods, or samples
    examined in the Market Basket Survey (Lipscomb, 1968).

        The only DDT exposures of infants that are known to have injured
    them in any way are those involving direct access to formulations that
    they ate or drank. Such tragic accidents often involved formulations
    transferred to unlabelled food or beverage containers. Frequently, the
    container was left where a child could reach it easily. Occasionally,
    formulations have been stored with food or have ever been handed to a
    child as food, or "empty" containers that still contain enough
    formulation to kill a child have been carelessly discarded (Hayes,
    1975, 1976a).

    5.3  Occupational Exposure

        In general, annual exposure to DDT is greatest among manufacturers
    and formulators, moderate, among those applying it for agricultural
    purposes, less among the general population, and least among special
    groups whose location or practices minimize their exposure. However,
    for brief intervals the exposure during agricultural application may
    exceed anything that good industrial practice permits. This
    distinction is of great importance in connexion with the more toxic
    organic phosphorus compounds, a single heavy exposure to which may
    result in poisoning, but is of no known importance in connexion with
    DDT, the acute toxicity of which is much less. However DDT has a
    greater tendency to storage in the body.

        Occupational exposure to DDT is reflected quantitatively by the
    concentration of DDT and DDE in blood and fat and by the concentration
    of DDA in urine. These aspects of occupational exposure are considered
    later. This section outlines measurements that have been made of the
    actual degree of exposure under different circumstances.

        The most striking result is that the occupational exposure through
    areas of skin that are frequently unclothed (face, hands, forearms,
    neck, and "V" of chest) is far greater than total respiratory
    exposure. Results for DDT are shown in Table 4 (based on measurements
    made by methods described by Durham & Wolfe (1962, 1963)). If a
    workman has bare feet or legs or does not wear a shirt, the contrast
    between respiratory and dermal exposure will be even greater than that
    shown in Table 4 which is based on "standard" clothing.

    Table 4.  Measured respiratory and dermal exposure of workers
              to DDT under actual conditions of work

    Activity                 Respiratory   Dermal     Reference
                             (mg/kg)       (mg/kg)

    Indoor house spraying    3.4a          1755       Wolfe et al., 1959
    Outdoor house spraying   0.11           243       Wolfe et al., 1959
    Spraying forests         4-92b          212b      Wassermann
                                                      et al., 1960

    a  Measurement by respirator pad technique.
    b  Calculated from values given in the original paper.

        It has been reported that DDT and a number of other pesticides
    persist, for days or even years after last use, on the hands of
    workers exposed to them and that at least a part of the material can
    be removed for analysis by rinsing the hands in hexane. Evidence that
    the residues represented unabsorbed pesticides and not excretion of
    stored material included the fact that no correlation was found
    between residues on the hands and in the sera of individuals and that
    no residues could be found on the hands of a farmer who used
    rubberized gloves while working with pesticides and who washed
    afterwards with strong cleansing agents (Kazen et al., 1974). The fact
    that urinary excretion of DDA may be increased for several days after
    a single occupational exposure to DDT (Wolfe et al., 1970) is evidence
    that absorption continues for a week or so but not for longer periods.
    Furthermore, Wolfe et al. (1970) found that the rate of excretion,
    even when detectable, was small compared to the excretion of parathion
    metabolite in workers exposed simultaneously to DDT and parathion at a
    ratio of 1.0 to 0.25. This emphasizes the minimal dermal absorption of
    DDT.

    6.  METABOLISM OF DDT

    6.1  Uptake

    6.1.1  Uptake by inhalation

        Most DDT dust is of such large particle size (>250 µm) that any
    that is inhaled is deposited in the upper respiratory tract and is
    eventually swallowed (Hayes, 1975). Toxicity data indicate that
    respiratory exposure is of no special importance.

    6.1.2  Uptake from the gastrointestinal tract

        A review of the early literature indicates that absorption of DDT
    from the gastrointestinal tract is slow. Whereas intravenous injection
    at the rate of 50 mg/kg produces convulsions in rats in 20 min,
    convulsions occur only after 2 h when DDT is administered orally at
    two or more times the LD50 value. The onset of convulsions is
    delayed for about 6 h when DDT is given to rats orally at
    approximately the LD50 value (Dale et al., 1963).

        Early studies based on toxicity indicated that DDT, dissolved in
    animal or vegetable fats, was absorbed from the gastrointestinal tract
    about 1.5 to 10 times more effectively than undissolved DDT. There was
    also evidence that large doses of the compound in the gastrointestinal
    tract were poorly absorbed from nonabsorbable solvent (Hayes, 1959).
    However, in connexion with small repeated doses, the presence or kind
    of solvent made little difference; apparently the occurrence of bile
    in the intestine and the presence of some fat in the diet were
    sufficient to promote absorption of the compound. At high dosage
    levels, less 14C-DDT was absorbed and stored in organs and a higher
    proportion was excreted in the faeces following oral administration
    than after intraperitoneal administration (40% versus 0.9%) (Bishara
    et al., 1972b).

        Rothe et al. (1957) reported that after giving radioactive DDT to
    rats by stomach tube, 41-57% of it was recorded in lymph drained from
    the animal by means of a cannula. Less than 0.1% of the activity was
    found in the urine, 7.4-37.1% was found in the faeces or in the
    intestinal contents when the animals were killed, and 19%-67% of the
    activity was found in the carcass. The total dose accounted for
    analytically varied from 89% to 118%, thus recovery was complete
    within the accuracy of the method. Of the administered DDT not found
    in faeces and intestinal contents, 47%-65% was found in the lymph. The
    animals that withstood the operation best had peak lymph flows of
    nearly 6 ml/h. In these animals, DDT was absorbed at rates as high as
    381 µg/h; the rate of absorption reached a maximum within 2-3 h of
    intubation and was markedly reduced by the fourth hour. Fifty per cent
    of the DDT-derived material found in the lymph was absorbed in the
    first 2.5-7 h, and 95% was absorbed within 18 h. Because the lymphatic

    duct in the rat is not a single vessel, Rothe et al. (1957) were
    unable to exclude the possibility that some, or all, of the DDT that
    they later recovered from the carcasses of their animals had been
    transported to the general circulation by collateral lymph vessels
    rather than by the hepatoportal system. Thus, at least half, and
    perhaps all absorption of DDT is by way of the lymph. However, in
    studies on rats by Heath & Vandekor (1964), only a small proportion of
    dieldrin was absorbed by the lymph. The reason for the marked
    difference in the absorption of those organochlorine insecticides is
    unknown.

    6.1.3  Uptake from the skin

        Undissolved DDT is so poorly absorbed through the skin that its
    toxicity by this route is difficult to measure. Even dissolved DDT is
    poorly absorbed by the skin as indicated by low toxicity (see
    Table 5).

    Table 5.  Acute oral and dermal LD50 of DDT for animalsa

    Species      Formulation                    Oral        Dermal
                                                (mg/kg)     (mg/kg)

    Rat          Water suspension or powder     500-2500    1 000 000
                 Oil solution                   113-450     250-3000

    Mouse        Water suspension or powder     300-1600    375 000
                 Oil solution                   100-800     250-500

    Guineapig    Water suspension or powder     2000        1 500 000
                 Oil solution                   250-560     1000

    Rabbit       Water suspension or powder     275         375 000
                 Oil solution                   300-1770    300-2820

    Cat          Water suspension or powder
                 Oil solution                   100-410

    Dog          Water suspension or powder
                 Oil solution                   >300

    a  From: Hayes (1959).

    6.2  Distribution and Storage

    6.2.1  Human studies

    6.2.1.1  Studies of volunteers

        In a study of volunteers who received technical DDT at rates of 0,
    3.5, and 35 mg/man per day, the average intakes resulting from dosing
    and from traces of DDT in food were 0.0025, 0.05, and 0.5 mg/kg per
    day (Hayes et al., 1956). The storage of DDT was proportional to
    dosage, but there was an unexplained difference in the storage of the
     p,p'-isomer and of technical DDT. For example, following dosing for
    12 months, pure  p,p'-DDT was stored in fat at an average
    concentration of 340 mg/kg, but the sum of isomers from technical
    material was stored at an average of only 234 mg/kg. The difference
    was statistically significant for the 3.5 mg/man per day dosages given
    for 3-6 and for 7-18 months. The difference was significant for the
    35 mg/man per day doses after 7-18 months of dosing but not after only
    3-6 months.

        Men who ate  p,p'-DDT showed a definite increase in the absolute
    amount of DDE stored. After 6 months at a dosage of 35 mg/man per day,
    8 men showed an average concentration of DDE stored in fat of 33 mg/kg
    compared with 12 mg/kg for the same individuals at the beginning of
    the investigation. There was a further increase in DDE storage as
    exposure progressed. However, DDT was stored in so much greater
    concentration that the relative storage of DDE decreased sharply.
    Thus, after 6 months at a dosage of 35 mg/man per day, 8 men stored
    only 14% of their total DDT-derived material in the form of DDE
    compared with 65% for the same person at the beginning of the
    investigation.

        The storage of DDE by men who ate technical DDT presented a
    different picture. Until 18 months after exposure, there was no clear
    evidence that these men stored any more DDE after exposure than they
    did before. However, at 18 months, the only 3 samples available showed
    DDE concentrations ranging from 28 to 85 mg/kg, all substantially
    above general population levels. Thus, both the total amount stored
    and the rate at which DDT converted to DDE served to distinguish the
    metabolism of  p,p'-DDT and the sum of isomers present in technical
    DDT in man (Hayes et al., 1956). A more rapid excretion was
    demonstrated for  o,p'-DDT by Morgan & Roan (1972).

        In a second study (Hayes et al., 1971), volunteers received the
    same doses used in the first study. Again, storage of DDT was
    proportional to dosage. Although, in this instance also, the storage
    of technical DDT was less than that of  p,p'-DDT, the difference was
    not statistically significant. The real but very gradual accumulation
    of DDE was confirmed.

        A steady state of storage was approached later in the second study
    (18.8-21.5 months) than in the earlier one (about 12 months). However,
    although the second study was superior in that more men were studied
    for a longer period, it was inferior in that dosage was less regular.
    Because of this, it seems impossible to decide whether 12 months or
    21.5 months is a more valid estimate of the time necessary for people
    to approach a steady state of storage when intake is uninterrupted and
    unvarying in amount. It is interesting that the storage levels
    eventually reached at the same dosage in the 2 studies were
    statistically indistinguishable in most instances (see Table 6). In
    the one instance in which a statistical difference existed, the
    greater storage by men in the second study may have been explained by
    the fact that some of them inadvertently received higher doses than
    intended.

        Table 6.  Storage of DDT in volunteers

    Type of DDT      Added      Concentration of DDTa                          Significance
                     dosage     First studyb            Second studyc          of difference
                     (mg/man    11 months or more       21.5 months             (P)
                     per day)   (mg/kg)                 (mg/kg)

    Technical           0       8-17 (12.5 ± 4.5)       16-30 (22.0 ± 2.9)     > 0.1
                        3.5     26-33 (23.8 ± 1.4)      59-76 (50.2 ± 5.6)     <0.025
                       35       101-367 (234 ± 21.4)    105-619 (281 ± 79.5)   >0.4

    Recrystallized     35       216-466 (340 ± 36.4)    129-659 (325 ± 62.2)   >0.2

    a  Range, mean, and standard error.
    b  Hayes et al., 1956,
    c  Hayes et al., 1971.

        There was a slow decrease in the levels of fat-stored DDT after
    dosing ceased. The concentration remaining following 25.5 months of
    recovery was from 32% to 35% of the maximum stored for those who had
    received 35 mg/man per day but 66% for those who had received only
    3.5 mg/man per day, indicating slower loss at lower storage levels
    (Hayes et al., 1971).

        Morgan & Roan (1971) fed volunteers not only technical DDT but
    also  p,p'-DDE and  p,p'-TDE. They found that DDE was stored more
    tenaciously than the other compounds in man, the order being  p,p'-DDE
    >  p,p'-DDT >  o,p'-DDT >  p,p'-TDE. The slow metabolism of DDT to
    DDE was confirmed. It was noted that  p,p'-DDT was lost from storage
    in adipose tissue much more slowly in man than in the monkey, dog, or
    rat.

        Less than 18% of  p,p'-DDT and  p,p'-DDE is carried in human
    erythrocytes. In plasma of ordinary fat content, less than 1% of all
    DDT-related compounds is carried by the chylomicrons. Instead, these
    compounds are carried by proteins and are undetectable in plasma from
    which protein has been precipitated. Following ultracentrifuging,
     p,p'-DDT and  p,p'-DDE are found mainly in the triglyceride-rich,
    low density, and very low density lipoproteins. Following continuous
    electrophoresis, these compounds are found mainly in association with
    plasma albumin and alpha-globulins (Morgan et al., 1972).

    6.2.1.2  Studies of occupationally exposed workers

        The highest reported storage of DDT and related compounds remains
    that of a healthy worker whose fat contained DDT and DDE (as DDT) at
    concentrations of 648 and 483 mg/kg, respectively (Hayes et al.,
    1956). Laws et al. (1967) reported considerably lower storage values
    among the most exposed persons in a DDT manufacturing plant (see
    Table 7).

        An important point evident from the table is that, whereas almost
    all investigations of workers are said to have been carried out on
    "heavily exposed" populations, some of the groups studied had absorbed
    little more DDT than is absorbed by the general population --
    especially the general population of some tropical countries.

        A different situation is indicated in a report by Genina et al.
    (1969) who used a total chloride method to analyse samples of blood
    from controls and from persons with occupational exposure to DDT,
    polychloropinene, and HCH. Whereas the highest average concentration
    of total DDT-related material  per se in the serum of a worker in the
    USA was 2.7 mg/litre (Laws et al., 1967), Genina et al. (1969)
    reported organochlorine compounds as high as 38.4 mg/litre in the
    blood of a pilot and values as high as 195 mg/litre in the blood of
    warehousemen. This concentration is about 20 times the highest value
    found by the same authors in their control group (see Table 8). The
    factor of 20 is not remarkable, but (especially in view of the fact
    that polychloropinene and HCH are excreted more readily than DDT and
    DDE) values as high as 0.2 mg/litre in the controls are unexpected.

        Table 7.  Average concentration of the sum of the isomers of DDT and DDE in fat and serum
              and of DDA in the urine of workers engaged in the manufacture, formulation, or
              use of DDT

    Tissue    No. of    DDT         DDE         DDA        Total as     Estimated    Reference
              men       (mg/kg)     (mg/kg)     (mg/kg)    DDT          exposure
                                                           (mg/kg)      (mg/man
                                                                        per day)

    fat          1      648         437                    1131                      Hayes et al., 1956
    urine       10                                0.57                    14         Ortelee, 1958
    urine       16                                1.7                     30         Ortelee, 1958
    urine       13                                2.9                     42         Ortelee, 1958
    fat          3       51          44                      98            3.6       Laws et al., 1967
    fat         12       74          50                     130            6.2       Laws et al., 1967
    fat         20      161          91                     263           18         Laws et al., 1967
    serum        3        0.2113      0.1968                  0.5412       6.3       Laws et al., 1967
    serum       12        0.1420      0.1454                  0.3584       8.4       Laws et al., 1967
    serum       20        0.3020      0.2719                  0.7371      17.5       Laws et al., 1967
    urine        3        0.0165      0.0203      0.41        0.5629                 Laws et al., 1967
    urine       12        0.0145      0.0222      0.6         0.7911                 Laws et al., 1967
    urine       20        0.0145      0.0271      1.27        1.6296                 Laws et al., 1967
    fat         18                                            5.2-45.2               Gracheva, 1969
    urine      136                                0.402                              Perini & Ghezzo, 1970
    urine      110                                0.142                              Perini & Ghezzo, 1970
    urine      290a                               0.061                              Perini & Ghezzo, 1970
    plasma      16        0.0513      0.0722                  0.1321                 Wassermann et al., 1970c
    serum        4       <0.087      <0.072                                          Edmundson et al., 1970a
    urine                                         0.080                              Edmundson et al., 1970a
    serum       18        0.573       0.506                                          Poland et al., 1970
    serum        5        0.004a      0.021a                                         Clifford & Well, 1972
    serum       10        0.022       0.055                                          Clifford & Well, 1972
    serum       21        0.021a      0.013a                                         Keil et al., 1972
    blood       44                                            0.761                  WHO, 1973
    blood      100                                            1.273                  WHO, 1973

    Table 7 (Cont'd)

    Tissue    No. of    DDT         DDE         DDA        Total as     Estimated    Reference
              men       (mg/kg)     (mg/kg)     (mg/kg)    DDT          exposure
                                                           (mg/kg)      (mg/man
                                                                        per day)

    serum       21        0.300       0.379                   0.681                  Almeida et al., 1974
    serum       25        0.225       0.308                   0.504                  Almeida et al., 1974
    serum       18        0.345       0.257                   0.602                  Almeida et al., 1974
    serum       56        0.004a,b    0.052a,b                                       Morgan & Roan, 1974
    serum       32        0.002b      0.026b                                         Morgan & Roan, 1974
    serum       32        0.004b      0.047b                                         Morgan & Roan, 1974
    serum       32        0.009b      0.075b                                         Morgan & Roan, 1974
    serum       31        0.052b      0.222b                                         Morgan & Roan, 1974
    plasma      25                                            1.030                  Rabello et al., 1975
    plasma       8                                            0.240                  Rabello et al., 1975
    plasma      23                                            0.0389                 Richardson et al., 1975

    a  Control group.
    b  Approximately equal groups arranged by degree of storage.

    Table 8.  Percentage of workers with blood organochlorine compound content falling in certain
              ranges of concentrationa

    Subject group         Range of concentrations
                                                                                                       
                          0             0.2-0.9       1.0-3.0      4-9          10-50         50
                          (mg/litre)    (mg/litre)    (mg/litre)   (mg/litre)   (mg/litre)    (mg/litre)

    Control group (47)    21.3          44.7          23.4         10.6         0             0
    Pilots (134)
      group A             19.1          35.3          25.0         14.7         5.9           0
      group B             22.9          27.1          40.6         7.4          2.0           0

    Technicians (133)
      group A             26.2          16.4          39.3         8.2          9.9           0
      group B             39.4          31.2          25.7         3.7          0             0

    Agricultural          13.0          43.5          31.0         7.0          2.0           3.5
    workers (55)

    a  From:  Genina et al. (1969).
    NB: Group A gives the results of investigations at the time of work, group B before work
    or a few months after termination. Agricultural workers were studied only during work.

        The first evidence that a part of the DDT absorbed by man is
    metabolized to DDE was obtained from the analysis of fat from a DDT
    plant worker (Mattson et al., 1953).

    6.2.1.3  Studies of the general population

         DDT in fat. Table 9 summarizes the results of measurements of
    DDT and related materials in the body fat of people without
    occupational exposure. Several broad generalizations can be made from
    the table and from what is known about residues of DDT in food in
    different countries. DDT storage in man corresponds with exposure; in
    general, exposure tends to be greater in warm climates where there is
    a greater need for insecticides. This climatic dependence may be
    observed even within a single country (Hayes, 1975). Where
    measurements have been made during a sufficiently long period, storage
    has decreased as the use of DDT, especially that leading to residues
    in food, has decreased.

        The method of DDT analysis shifted in about 1962 from the
    Schecter-Haller colorimetric method to the gas chromatographic method.
    However, as noted by Hayes (1975), this made little difference to the
    overall results. Thus the absolute decrease of total DDT storage and
    the relative increase of DDE storage observed in some countries is
    real.

        Circumstances peculiar to some subpopulations explain their
    unusual storage of DDT. Thus, persons living in one of the contiguous
    states of the USA stored significantly less DDT than other persons in
    the state because they did not eat meat (Hayes et al., 1958). Even
    less storage of DDT was observed in Eskimos whose diet contained an
    unusually high proportion of meat obtained from wildlife in which no
    DDT and almost no DDE could be detected (Durham et al., 1961).

        No significant difference was found in the concentration of DDT
    stored in the fat of different parts of the body (Hayes et al., 1958;
    Casarett et al., 1968).

         DDE in fat. The accumulation of DDE relative to total DDT-related
    compounds is best illustrated in man. Of the total DDT stored
    in the fat of workers exposed to technical DDT (about 4% DDE) for
    11-19 years, only 38% was in the form of DDE, and, of course, some of
    that DDE came from their diets which included meat (Laws et al.,
    1967). In India, where many people avoid meat but may consume milk,
    cheese, and eggs, 34-41% of total DDT was DDE (Dale et al., 1965). In
    the USA, during a time when DDT residues in food were decreasing, the
    proportion of total DDT in the form of DDE increased from about 60% in
    1955 to about 80% in 1970; during the same interval the concentration
    of total DDT in body fat decreased from about 15 mg/kg to slightly

        Table 9.  Concentration of DDT-derived material in body fat of the general population

    Country            Year            No. of    Method of   DDTa      DDE as    Total as     DDE as  Reference
                                       samples   analysis    (mg/kg)   DDT       DDT          DDT
                                                                       (mg/kg)   (mg/kg)      (% of
                                                                                              total)

     North America
    Canada             1959-1960         62      colour        1.6       3.3        4.9        67     Read & McKinley, 1961
    Canada             1966              47      GLCb and ELC  1.09      2.96       4.39       67     Brown, 1967
    Canada             1967-1968         51      GLC           1.56      4.16       5.86       71     Kadis et al., 1970
    Canada             1969                                                         4.85              Ritcey et al., 1973
    Canada                                       GLC                                5.83              Brown & Chow, 1975
    USA                1942              10      colour      NDc       NDc        NDc                 Hayes et al., 1958
    USA                1950              75      colour        5.3      --          5.3        --     Laug et al., 1951
    USA                1955              49      colour        7.4      12.5       19.9        63     Hayes et al., 1956
    USA                1954-1956         61      colour        4.9       6.8       11.7        58     Hayes et al., 1958
    USA                1956              36      colour        5.5      10.1       15.6        65     Hayes et al.. 1971
    USA                1961-1962        130      colour        4.0       8.7       12.7        69     Quinby et al., 1965a
    USA                1961-1962         28d     GLC           2.4       4.3        6.7        64     Dale & Quinby, 1963
    USA                1962-1963        282      GLC           2.9       8.2       11.1        74     Hoffman et al., 1964
    USA                1964              64      GLC           2.5       5.1        7.6        67     Zavon et al., 1965
    USA                1964              25      GLC           2.3       8.0       10.3        77     Hayes et al., 1965
    USA                1964-1965         18      GLC                                9.0               Schafer & Campbell, 1966
    USA                1964-1965         42      GLC           3.1       7.5       10.6        71     Radomski et al., 1968
    USA                1962-1966        994      GLC           2.6       7.8       10.4        75     Hoffman et al., 1967
    USA                1964-1965         12      GLC           3.79      7.7       11.5        67     Davies et al.. 1965
    USA                1965-1967         17      GLC                     3.1        5.5        56     Davies et al., 1968
    USA                                  90      GLC                     6.1        8.4        73     Davies et al., 1968
    USA                                  17      GLC                     4.6        7.8        59     Davies et al., 1968
    USA                                  35      GLC                    12.0       16.7        72     Davies et al., 1968
    USA                --                42      GLC           3.13e     7.43      10.56       70     Fiserova-Bergerova et
                                                                                                      al., 1967

    Table 9 (Cont'd)

    Country            Year            No. of    Method of   DDTa      DDE as    Total as     DDE as  Reference
                                       samples   analysis    (mg/kg)   DDT       DDT          DDT
                                                                       (mg/kg)   (mg/kg)      (% of
                                                                                              total)

    USA                --                30e     GLC           1.33      5.17       6.51       79     Casarett et al., 1968
    USA                                  29f     GLC           1.35      4.91       6.31       78     Casarett et al., 1968
    USA                                  30g     GLC           1.16      4.99       6.17       81     Casarett et al., 1968
    USA                1966-1968         70      GLC           1.54      5.15       6.69       77     Morgan & Roan, 1970
    USA                1967             733      GLC           1.34      4.74       6.22       77     Yobs, 1969 (unpublished
                                                                                                      data)
    USA                1968            3104      GLC           1.56      5.96       7.67       77     Yobs, 1969 (unpublished
                                                                                                      data)
    USA                1967-1971        103      GLC           1.5       5.6        7.1        79     Warnick, 1972
    USA                1970             200      GLC           1.9       8.0        9.9        81     Wyllie et al., 1972
    USA                1969-1972        221                                        23.18              Burns, 1974
    USA                1970            1412                                         7.87h,i           Kutz et al., 1974

     South America
    Argentina          1967              37      GLC           5.5       6.5       13.2        --     Wassermann et al., 1968b
    Brazil             1969-1970         38      GLC           1.4       2.7        4.1               Wassermann et al., 1972b
    Venezuela          1964              38      GLC           2.9       7.4       10.3        72     Dale, 1971 (unpublished
                                                                                                      data)

     Europe
    Austria                                                                         6.33              Pesendorfer et al., 1973
    Belgium                              20      GLC           1.2       2.1        3.3        64     Maes & Heyndrickx, 1966
    Bulgaria                             55      GLC           3.8       5.8       10.6n       57     Kaloyanova et al., 1972
    Bulgaria           1971-1976        191      GLC                               14.7        78     Rizov, 1977
    Czechoslovakia     1963-1964        229      colour        5.5       4.1        9.6        43     Halacka et al., 1965
    Denmark            1965              18      GLC           0.6       2.7        3.3        82     Weihe, 1966
    Denmark            1972-1973         78      GLC                     4.1        4.7        87     Kraul & Karloq, 1976

    Table 9 (Cont'd)

    Country            Year            No. of    Method of   DDTa      DDE as    Total as     DDE as  Reference
                                       samples   analysis    (mg/kg)   DDT       DDT          DDT
                                                                       (mg/kg)   (mg/kg)      (% of
                                                                                              total)

    Finland            1972-1974         73      GLC                                2.5               Hattula et al., 1976
    France             1961              10      colour        1.7       3.5        5.2        67     Hayes et al., 1963
    German Democratic  1966-1967        100      GLC and TLC   3.7       9.47      13.1        71     Engst et al., 1967
    Republic
    German Democratic                    34j                   1.8       5.1        6.9               Engst et al., 1970
    Republic
    Germany, Federal   1958-1959         60      colour        1.0       1.3        2.3        57     Maier-Bode, 1960
    Republic of
    Germany, Federal   1970              20      GLC           1.1       2.5        3.6        69     Acker & Schulte, 1970
    Republic of
    Germany, Federal                                                                9.8               Acker & Schulte, 1971
    Republic of
    Germany, Federal                                                                4.24              Acker & Schulte, 1974
    Republic of
    Germany, Federal                                                                4.77              Acker & Schulte, 1974
    Republic of
    Germany, Federal                                                                5.42              Acker & Schulte, 1974
    Republic of
    Germany, Federal                                                                8.36              Acker & Schulte, 1974
    Republic of
    Germany, Federal                                                                7.8               Acker & Schulte, 1974
    Republic of
    Hungary            1960              48      colour        5.7       6.0       12.4        48     Denés, 1962
    Italy              1965               9      GLC           1.8       3.2        5.0        63     Kanitz & Castello, 1966
    Italy              1965-1966         18      GLC           2.58      8.28      10.86       76     Paccagnella et al., 1967
    Italy              1966              22      GLC and TLC   4.69     10.69      15.48       68     Del Vecchio & Leoni, 1967
    Italy              1970?             31      GLC           3.38     13.37      16.75       80     Prati & Del Dot, 1971
    Italy              1970?             52      GLC           2.14a     8.46a     10.60a      80     Prati et al., 1972
    Italy              1970?             33      GLC           0.84a     6.64a      7.48a      89     Prati et al., 1972

    Table 9 (Cont'd)

    Country            Year            No. of    Method of   DDTa      DDE as    Total as     DDE as  Reference
                                       samples   analysis    (mg/kg)   DDT       DDT          DDT
                                                                       (mg/kg)   (mg/kg)      (% of
                                                                                              total)

    Netherlands        1964              20      colour        1.6       6.1        7.7        79     Wit, 1964
    Netherlands        --                11      GLC           0.32      1.89       2.22       86     deVleiger et al., 1968
    Norway                               56      GLC                                3.2        71     Bjerk, 1972
    Poland             1965              72      colour                            13.4               Bronisz st al., 1967
    Poland                               65      colour                            23.5        62     Bronisz et al.. 1969
    Poland             1970              70      GLC                               11.4               Juszkiewicz & Stec, 1971
    Poland             1972              15      GLC                                5.23       68     Bojanowska et al., 1973
    Romania            1965             137      --           13.4       8.3       21.7        39     Aizicovici et al., 1968
    Romania            1972-1973                 GLC           0.68      1.73       2.41       71     Ciupe, 1976
    Spain              1966              41      --            6.5       9.2       15.7        59     Unpublished data cited
                                                                                                      by Wassermann et al., 1968
    Switzerland                                                                     1.9-16.3          Zimmerli & Marek, 1973
    United Kingdom     1961-1962        131      colour       --        --          2.2        --     Hunter et al., 1963
    United Kingdom     1963-1964         66      GLC           1.1       2.2        3.3h       67     Egan et al., 1965
    United Kingdom     1964             100      GLC          --        --          3.9h       --     Robinson et al., 1965
    United Kingdom     1964              44      GLC          --        --          4.0h       --     Robinson & Hunter, 1966
    United Kingdom     1965             101      GLC           1.13      1.72       2.85       60     Cassidy et al.. 1967
    United Kingdom     1965-1967        248      GLC           0.78      2.22       3.00       74     Abbott et al., 1968
    United Kingdom     1969-1971        201      GLC           0.5       1.8        2.5        72     Abbott et al., 1972
    USSR                                 41      TLC           4.33      3.73                         Vas'kovskaja & Komarova.
                                                                                                      1967
    USSR                                197      TLC           5.3-      3.2-                         Vas'kovskaja, 1969
                                                               7.57      7.1

     Africa
    Kenya                                                                           5.4               Wassermann et al., 1972a
    Nigeria            1967              43      GLC           5.4       3.1        8.8               Wassermann et al., 1968b
    Nigeria            1969              41      GLC           2.1       2.8        6.5        57     Wassermann et al., 1972d

    Table 9 (Cont'd)

    Country            Year            No. of    Method of   DDTa      DDE as    Total as     DDE as  Reference
                                       samples   analysis    (mg/kg)   DDT       DDT          DDT
                                                                       (mg/kg)   (mg/kg)      (% of
                                                                                              total)

     Africa
    South Africa                                                                    5.94       43     Wassermann et al., 1970b
    South Africa                                                                    7.16              Wassermann et al., 1970b
    Uganda                                                                          2.9               Wassermann et al., 1974a

     Asia
    India (Delhi area, 1964              67      colour       17        10         26          39     Dale et al., 1965
    civilian)
    India (other       1964              16      colour        8         5         13          37     Dale et al., 1965
    cities, military)
    India                                94      GLC                               13.8h       42     Ramachandran et al., 1974
    Israel             1963-1964        254      colour        8.5      10.7       19.2        56     Wassermann et al., 1965
    Israel             1965-1966         71      colour        4.6                                    Wassermann et al., 1967
    Israel             1965-1966        133      colour        8.2                                    Wassermann et al., 1967
    Israel             1967-1971         63      GLC           3.0      10.6       14.4        74     Wassermann et al., 1974b
    Japan              1968-1969        241      GLC           0.6       1.8        2.4        75     Curlay et al., 1973
    Japan              1969-1970         74                                         6.92              Nishimoto et al., 1970
    Japan              1970                                                         4.499             Suzuki et al., 1973
    Japan              1971                                                         2.694             Suzuki et al., 1973
    Japan              1971              30                                        12.895             Kasai st al., 1972
    Japan              1972                                                         4.001             Suzuki et al., 1973
    Japan              1972              42                                         5.992             Kasai et al., 1972
    Japan              1973                                                         6.44              Kawanishi et al., 1973
    Japan              1974                                                         6.87              Inoue et al., 1974
    Pakistan                             60                                        25.0               Mughal & Rahman, 1973
    Thailand           1969-1970         77                                        12.6               Wassermann et al., 1972c

     Oceania
    Australia          1965              53      GLC           0.77h     1.03h      1.81h      57     Bick, 1967
    Australia          1965-1966         46      colour        3.6       6.6       10.2        64     Wassermann et al., 1968a
    Australia          1965-1966         12      GLC           3.0       7.1       10.5        68     Wassermann et al., 1968a
    Australia          1971              75      GLC                                4.94              Brady & Slyali, 1972

    Table 9 (Cont'd)

    Country            Year            No. of    Method of   DDTa      DDE as    Total as     DDE as  Reference
                                       samples   analysis    (mg/kg)   DDT       DDT          DDT
                                                                       (mg/kg)   (mg/kg)      (% of
                                                                                              total)

     Oceania
    New Zealand        1966              52      GLC           1.6       4.2        5.8        72     Brewerton & McGrath, 1967
    New Zealand        1965-1969        254      GLC           3.5      11.0       14.6        75     Copplestone et al., 1973

    a   p,p'-DDT and  o,p'-DDT only. Total as DDT includes TDE (DDD) and other forms when given, which are not shown in this table.
    b  Gas-liquid chromatography.
    c  Not detected,
    d  These 28 samples were also tested for DDT and DDE content by a colorimetric method, and the results are included in the
       130 samples listed above.
    e  Perirenal fat.
    f  Mesenteric fat.
    g  Panniculus fat.
    h  Geometric mean.
    i  Lipid basis.
    j  Infants and children.

    less than 10 mg/kg (see Table 9). Thus, a low proportion of DDE
    indicates a relatively high intake of DDT, a relatively low intake of
    DDE residues (e.g. in food), and relatively few years for the
    metabolism of stored DDT to DDE.

         DDT in blood. The finding of DDT and related compounds in blood
    (usually serum) of the general population of different countries is
    shown in Table 10. Compared to body fat, blood has been analysed for
    DDT in fewer countries and for fewer years. Some of the same
    differences between populations and time periods observed in connexion
    with DDT in fat have been detected in connexion with DDT in blood, but
    the differences are less marked.

        There is a small but statistically significant decrease in the
    concentrations of DDT-related compounds in the plasma of women 1-6
    days postpartum compared with the same women early in pregnancy. Most
    of the decrease seems to occur during about the last 10 days before
    delivery (Curley & Kimbrough, 1969). In a similar way, the
    concentration of DDT-related compounds in various tissues of women at
    the time of Caesarean section or normal delivery is less than in
    nonpregnant women in the same community (Polishuk et al., 1970).

        The concentration of  p,p'-DDE is remarkably constant throughout
    the day, but minor increases in it and in  p,p'-DDT may occur after a
    meal (Radamski et al., 1971a).

        In so far as can be judged from the bar graphs presented by
    Griffith & Blanke (1975), the concentrations of DDT-related materials
    they found in postmortem blood were similar to the concentrations
    reported in Table 10 for fresh blood except that postmortem blood
    contained more TDE (DDD).

         DDT and related compounds in other tissues. DDT has been found
    in some samples of all human organs. Results reported in the USA and
    USSR are shown in Tables 11 and 12, respectively. Results in Table 11
    are based on the wet weight of the tissues. Results in Table 12,
    presumably, are based on the weight of extractable lipid, but the
    paper does not specify this; whatever the basis, the paper reports
    concentrations of DDT and DDE in the range of 10.0-35.0 mg/kg in the
    heart muscle, liver, and kidneys of 2 persons who had had direct
    contact with pesticides. Although concentrations of chlorinated
    compounds in liver, kidney, brain, and spleen of persons from Ferrara
    Province, Italy, were generally higher than those in the USA (Prati et
    al., 1972), the differences were not great.

        Table 10.  Concentration mg/litre of DDT-related compounds in the blood of members of the general population

    Country         Year      No. of   p,p'-DDT   o,p'-DDT  p,p'-DDE  o,p'-DDE  p,p-    o,p-     Total DOT     DDE     Reference
                              samples                                           TDE     TDE      equivalent    as %
                                                                                (DDD)   (DDD)                  total

     North America

    Canada                                                                                       0.032                 Brown & Chow, 1975
    USA (Atlanta)    1965         10    0.0119     0.0013    0.0257                              0.0418         68.6   Dale et al., 1966b
    USA (Atlanta)    1966         10m                                                            0.0746                Dale et al., 1967
    USA (Atlanta)    1966         10f                                                            0.0360                Dale et al., 1967
    USA (Louisiana)  1966-1967    53a   0.00182    0.00182   0.00265  0.00265  0.00062  0.00062  0.00501        59.0   Selby et al., 1969
    USA (4 states)   1967         64    0.00335    0.00044   0.00837  0.00096  0.00032  0.00032  0.01425        78.0   Yobs, 1969
                                                                                                                       (unpublished data)
    USA (3 states)   1968        106    0.00342    0.00006   0.00927  0.00000  0.00004  0.00009  0.01397        73.4   Yobs, 1969
                                                                                                                       (unpublished data)
    USA (Idaho)      1967-1968  1000    0.0047               0.0220            0.0002            0.02940        83.5   Watson et al., 1970
    USA (Atlanta)    1969         30b,c 0.0046     0.0011    0.0062   0.0003   0.0014            0.0144         50.0   Curley et al., 1969
    USA              1968          5d                                                            0.0050                Curley & Kimbrough, 1969
    USA              1968         10d                                                            0.0205                Curley & Kimbrough, 1969
    USA (Florida)                 45e                        0.0108                                                    O'Leary et al., 1970b
    USA (Florida)                107g                        0.0152                                                    O'Leary et al., 1970b
    USA(Florida)     1970         26                                                             0.03169               Radomski et al., 1971a

     South America

    Argentina        1970         20h                                                            0.01934               Radomski et al., 1971b
                                  18i                                                            0.01327               Radomski et al., 1971b
                                  19j                                                            0.00869               Radomski et al., 1971b
    Brazil           1973?        15f   0.0189               0.0237                              0.0453                Schvartsman et al., 1974
    Brazil           1973?        15c   0.0118               0.0104                              0.0234                Schvartsman et al., 1974
    Brazil           1974?        30    0.145      0.026     0.155                               0.336                 Almeida et al., 1975
    Brazil           1974?        11    0.083                0.117                               0.194                 Almeida et al., 1975
    Brazil           1974?        20    0.086                0.121                               0.212                 Almeida et al., 1975

    Table 10 (Cont'd)

    Country         Year      No. of   p,p'-DDT   o,p'-DDT  p,p'-DDE  o,p'-DDE  p,p-    o,p-     Total DOT     DDE     Reference
                              samples                                           TDE     TDE      equivalent    as %
                                                                                (DDD)   (DDD)                  total

     Europe

    Bulgaria         1971-1976   171                                                             0.039a                Rizov, 1977
    Hungary          1967-1968   120                                                             0.034                 Czeglédi-Janko, 1969
    Poland                                                                                       0.172d                Jonczyk, 1970
    Poland           1972         15                                                             0.030          63     Bojanowska et al., 1973
    Switzerland                   13                                                             0.0209                Zimmerli & Marek, 1973

     Asia

    Israel           1975         29    0.0133     0.0112    0.0195   0.0112   0.0087   0.0072   0.0740         47     Polishuk et al., 1977
    Japan            1970         10                                                             0.011                 Tokutsu et al., 1970
    Japan            1971                                                                        0.005                 Kojima et al., 1971
    Japan            1971        138                                                             0.0183                Kasai et al., 1972
    Japan            1971                                                                        0.0093                Yamagishi et al., 1972
    Japan            1971                                                                        0.0285                Kaku, 1973
    Japan            1972                                                                        0.001-0.078           Study Group, 1972
    Japan                         37                                                             0.0437d               Nawa, 1973
    Japank                                                                                       0.1358                Hara et al., 1973
    Japanc                                                                                       0.0210                Hara et al., 1973
                                                                                                 0.0779                Abe et al., 1974
     Oceania

    Australia                     52                                                             0.0172                Slyali, 1972
    Australia                     47                                                             0.0169                Ouw & Shandar, 1974

    a  Geometric mean.                 d  Maximal value.   g  Black women.      j  1-5 years old.
    b  Mean of positive values only.   e  White women.     h  Adults.           k  Maternal blood.
    c  Cord blood from term infants.   f  Female.          i  6-11 years old.   m  Mile.

    Table 11.  Average concentrationsb of organochlorine insecticides in various tissues from autopsies
               of 44 members of the general populationa

    Tissue           No.         Lipid      DDT       DDE       TDE       Heptachlor  Dieldrin  Total + SEc
                     analysed    content    (mg/kg)   (mg/kg)   (DDD)     epoxide     (mg/kg)   (mg/kg)
                                 (%)                            (mg/kg)   (mg/kg)

    Perirenal fat       30       55.7       1.33      4.64      0.0110     0.0220     0.0300    6.03    ± 5.30
    Mesenteric fat      29       54.2       1.35      4.40      0.0470     0.0320     0.0630    5.89    ± 4.98
    Panniculus fat      30       60.6       1.16      4.48      0.0180     0.0270     0.0270    5.71    ± 5.25
    Bone marrow         19       20.6       0.411     2.08      0.0760     0.0040     0.620     2.63    ± 2.21
    Lymph noded         11        8.6       0.892     1.38      0.0100     0.0001     0.0190    2.30    ± 4.52
    Adrenal             18       10.5       0.125     0.875     0.0570     0.0012     0.0060    1.06    ± 1.31
    Kidney              38        3.2       0.0827    0.209     0.0022     0.0009     0.0056    0.300   ± 0.651
    Liver               42        2.1       0.0467    0.200     0.0326     0.0019     0.0037    0.285   ± 0.369
    Brain               32        7.9       0.0105    0.0831    0.0020     0.0002     0.0031    0.989   ± 0.171
    Gonad               36        1.3       0.0150    0.0688    0.0015     0.0001     0.0021    0.0875  ± 0.103
    Lung                25        0.7       0.0147    0.0585    0.0009     0.0003     0.0022    0.0766  ± 0.125
    Spleen              27        0.6       0.0112    0.0305    0.0031     trace      0.0021    0.0469  ± 0.074

    a  From: Cassarett et al. (1968).
    b  Wet tissue basis.
    c  SE = standard error of the mean.
    d  Tracheobronchial lymph nodes.

    Table 12.  Average DDT and DDE contents in certain human organsa

    Organ           Content      % of positive   DDT       DDE
                    limits       observations    (mg/kg)   (mg/kg)

    Heart muscle    1.0-12.0         77.0         3.69      4.05
    Liver           1.0-20.0         85.5         4.62      4.02
    Kidney          1.0-12.0         71.0         2.99      3.44
    Spleen          2.0-20.0         57.0         2.86      2.86
    Pancreas        2.0-15.0         75.0         3.0       2.62
    Adrenal         2.0-10.0         84.3         3.84      3.7
    Thyroid         2.0-8.0          59.0         1.85      1.85

    a From: Vas'kovskaja (1969).

         DDT and related compounds in the tissues of infants. The
    transfer of DDT to the fetus was observed by Deniés (1962) and
    confirmed by many others (Curley et al., 1969; Zavon et al., 1969;
    Komarova, 1970; O'Leary et al., 1970a,b,c). Typical findings are shown
    in Table 13. It appears that the placenta is partially protective; the
    concentrations of DDT and DDE are lower in cord blood than in
    corresponding maternal blood (O'Leary et al., 1970b; Schvartsman et
    al., 1974). The report by Engst et al. (1970) that infants lose a part
    of their DDT stores during the first few months of life, as a result
    of rapid growth, is not necessarily contradictory to the observation
    that at birth they have less than their mothers, but it must be said
    that the values reported by Engst et al. (1970) were relatively high
    for infants (see also section 5.2).

         Storage in relation to disease. A review (Hayes, 1975) indicates
    that there is no agreement in the literature regarding the effect of
    health on the storage of chlorinated hydrocarbon insecticides. Some
    investigators have not found any difference in the concentration of
    DDT in adipose tissue taken by biopsy or during minor elective surgery
    in contrast to that taken at autopsy. Some investigators, who used
    only autopsy samples, found no relationship between storage of
    chlorinated hydrocarbon insecticides and the cause of death. Others,
    of whom the first was Deichmann (Deichmann & Radomski, 1968; Radomski
    et al., 1968), have reported that storage was 1.7-7.6 times greater in
    persons dying of cirrhosis, atherosclerosis, hypertension, idiopathic
    amyloidosis, and certain forms of cancer. Weight loss was not really
    ruled out in these studies. Its importance was emphasized by Casarett
    et al. (1968), who found that disease did not influence concentrations
    on a wet weight basis but only on a lipid basis; samples with the
    highest levels of DDT on a lipid basis came from persons who not only
    had cancer but who were emaciated and had widespread abnormality of

        Table 13.  The range and mean of measurable concentrations of various organochlorines (mg/kg) in different tissues from stillborn infantsa

    Tissues                  p,p'-DDT    o,p'-DDT    p,p'-DDE    o,p'-DDE  p,p'-TDE    alpha-HCH   ß-HCH       gamma-HCH   Heptachlor   Dieldrin
    received                                                               (DDD)

    10       measurable      3           4           8           0         6           3           6           3           4            3
    adipose  concentration
             range           0.16-2.15   0.35-11.47  0.16-3.19   --        0.23-14.17  0.09-0.24   0.14-0.44   0.09-0.14   0.07-0.51    0.09-0.35
             mean            0.88        3.39        1.22        --        3.17        0.14        0.26        0.11        0.32         0.24
             SE ±            0.63        2.70        0.38        --        2.22        0.05        0.05        0.02        0.10         0.08

    8        measurable      1           1           2           0         2           0           1           1           0            1
    spinal   concentration
    cord     range           0.47        0.47        0.30-1.16   --        0.31-0.70   --          0.17        0.10        --           0.09
             mean            --          --          0.73        --        0.51        --          --          --          --           --
             SE ±            --          --          0.43        --        0.20        --          --          --          --           --

    8        measurable      3           1           4           0         3           3           1           1           1            2
    brain    concentration
             range           0.28-0.99   0.84        0.25-1.47   --        0.20-1.22   0.04-0.49   3.81        0.06        0.13         0.84-0.86
             mean            0.56        --          0.65        --        0.64        0.19        --          --          --           0.05
             SE ±            0.22        --          0.28        --        0.30        0.15        --          --          --           0.01

    9        measurable      3           2           6           0         3           2           3           3           2            1
    adrenals concentration
             range           1.28-1.65   0.36-1.05   0.13-1.96   --        0.91-1.45   0.40-0.57   0.12-0.71   0.20-0.53   0.46-1.00    0.92
             mean            1.48        0.71        1.05        --        1.11        0.49        0.37        0.33        0.73         --
             SE ±            0.11        0.35        0.28        --        0.17        0.09        0.18        0.10        0.27         --

    10       measurable      4           0           6           0         5           5           3           3           3            2
    lungs    concentration
             range           0.57-1.01   --          0.25-1.35   --        0.31-1.05   0.07-0.69   0.05-0.18   0.05-0.25   0.08-0.31    0.27-0.72
             mean            0.79        --          0.85        --        0.75        0.25        0.12        0.12        0.17         0.50
             SE ±            0.11        --          0.22        --        0.13        0.11        0.04        0.07        0.07         0.23

    Table 13 (Cont'd)

    Tissues                  p,p'-DDT    o,p'-DDT    p,p'-DDE    o,p'-DDE  p,p'-TDE    alpha-HCH   ß-HCH       gamma-HCH   Heptachlor   Dieldrin
    received                                                               (DDD)

    10       measurable      4           2           4           0         4           3           3           3           4            3
    heart    concentration
             range           1.04-4.17   0.57-0.68   1.27-4.79   --        1.02-5.82   0.23-0.52   0.15-0.31   0.19-0.27   0.30-1.56    0.08-1.02
             mean            2.17        0.63        2.74        --        3.27        0.33        0.22        0.22        0.80         0.49
             SE ±            0.69        0.06        0.74        --        1.10        0.09        0.05        0.03        0.30         0.28

    10       measurable      5           3           6           0         6           3           4           4           3            2
    liver    concentration
             range           0.15-1.59   0.22-3.42   0.22-2.45   --        0.19-2.14   0.21-0.32   0.03-0.20   0.05-0.36   0.03-1.67    0.16-0.22
             mean            0.79        1.32        0.98        --        1.01        0.25        0.11        0.24        0.68         0.19
             SE ±            0.24        1.05        0.34        --        0.29        0.04        0.04        0.07        0.50         0.03

    9        measurable      4           3           6           0         5           4           5           4           3            3
    kidney   concentration
             range           0.62-7.60   0.29-2.07   0.11-9.78   --        0.48-9.12   0.11-1.45   0.06-0.61   0.11-0.69   0.19-1.14    0.06-0.50
             mean            3.71        1.38        3.57        --        3.84        0.82        0.29        0.39        0.70         0.34
             SE ±            1.70        0.55        1.72        --        1.87        0.32        0.12        0.14        0.28         0.14

    8        measurable      3           2           3           1         3           1           1           2           5            3
    spleen   concentration
             range           0.48-1.04   0.45-2.94   0.60-1.05   0.29      0.18-0.91   0.21        0.16        0.17-0.18   0.10-0.52    0.18-0.51
             mean            0.80        1.70        0.86        --        0.56        --          --          0.18        0.35         0.31
             SE ±            0.17        1.25        0.13        --        0.21        --          --          0.005       0.08         0.10

    3        measurable      1           0           1           1         0           0           0           0           0            0
    pancreas concentration
             range           0.49        --          0.23        0.08
             mean            --          --          --          --        --          --          --          --          --           --
             SE ±            --          --          --          --        --          --          --          --          --           --
                                                                                                                                                
    a  From: Curley et al. (1969).

    the liver. Factors other than emaciation may be important in some
    conditions. For example, Oloffs et al. (1974) found that the DDT
    concentration in fat was not influenced by cirrhosis, but that the DDT
    concentrations in liver were significantly higher in cirrhotic livers
    with severe fatty infiltration and significantly lower in those with
    marked fibrosis or bile stasis.

        That increased storage of DDT is a result and not a cause of the
    diseases in which it sometimes occurs is shown by the fact that
    persons with extensive occupational exposure average 10 times more
    storage than the highest values reported in connexion with disease.
    However, they do not exhibit any predisposition to the diseases in
    question, and these diseases have shown no age-specific increase in
    incidence related to the introduction and use of insecticides.

    6.2.2  Animal studies

        A detailed review of the literature (Hayes, 1959) shows that a
    number of facts about the distribution and storage of DDT were
    established early either by single, classical papers (now fully
    confirmed) or by correlation of contributions from several
    laboratories. The major results may be summarized as follows:

        (a) DDT is stored in all tissues. Storage of the compound in
            blood, liver, kidney, heart, and the central nervous system
            was reported by Smith & Stohlman (1944).

        (b) Higher concentrations of DDT are usually found in adipose
            tissue than in other tissues (Ofner & Calvery, 1945).

        (c) Rats store DDT in their fat at all accurately measurable
            dietary levels including the unintended residues in standard
            laboratory feeds.

        (d) Following repeated doses, storage in the fat increases rapidly
            at first and then more gradually until a peak or plateau is
            reached (Laug et al., 1950). It was recognized that repeated
            doses at a moderate rate could result in greater total storage
            of DDT in the fat than a single dose at the highest rate that
            can be tolerated or even a single dose at a rate that
            frequently is fatal.

        (e) By plotting animal data published no later than 1950, it is
            possible to show that, when other factors are kept constant,
            the equilibrium storage of DDT in each tissue varies directly
            with the daily dosage.

        (f) However (with the apparent exception of the dog) storage in
            the fat, and perhaps in other tissues, is less extensive in
            relation to dosage at higher dietary levels (Fig. 1).

        (g) The rat, apparently, tends to lose a part of the DDT it has
            stored in fat at a peak level, reached in about 6 months, even
            though the same diet is continued.

        (h) There is a measurable, although not great, difference between
            the storage pattern of different species (Fig. 1).

        (i) At higher dosage levels but not at ordinary residue levels,
            the female rat consistently stores more DDT in its fat than
            the male, when offered the same diet (Fig. 1). The difference
            is only accounted for in part by the greater food intake of
            the female and must depend to some extent on more rapid
            biotransformation in the male. Other species show little or no
            sex difference.

        (j) The amount of DDT stored in the tissues gradually diminishes
            if exposure to the compound is discontinued or reduced.

        It is interesting to note that, even in the early studies, there
    was satisfactory agreement between different authors and, in fact,
    between different laboratories. Later studies have amplified some of
    the findings.

        Adams et al. (1974) observed that about the same concentrations of
    DDT and related compounds are stored by male rats and by females that
    reproduce successfully. The lower storage in mated females probably is
    accounted for by transfer to the young via the placenta and the milk.
    However, other factors may be involved; the disposal of the increased
    DDT taken in by the female rat as a result of her high food intake
    during lactation has not really been accounted for.

        When DDT, some of its analogues, and several other organochlorine
    insecticides were fed to male and female rats for 4 generations, there
    was little variation in storage of the materials, from one generation
    to another, and no evidence of a continuing increase in succeeding
    generations (Adams et al., 1974).

    Distribution to the fetus

        In animals, the concentrations of DDT in the blood and other
    tissues of the fetus are lower than those in the corresponding tissues
    of the mother (Dedek & Schmidt, 1972). The same is true in man (see
    section 5.2).

    Interaction with other compounds

        There was no evidence from studies on rats that dieldrin
    influenced the storage of DDT and its metabolites, even though DDT
    caused a marked reduction in storage of dieldrin (Street, 1964).
    However, Deichmann et al. (1971a) reported that the administration of

    capsules of aldrin (which is metabolized to dieldrin) to male and
    female dogs that had reached a steady state of DDT storage caused a
    rapid and progressive increase in the concentration of DDT, DDE, and
    TDE (DDD) in their blood and fat. The dosage of DDT was 12 (mg/kg)day
    and that of aldrin was 0.3 (mg/kg)day. Control dogs that continued to
    receive DDT but never received aldrin maintained a plateau of DDT
    storage. It is not clear whether the different results in rats and
    dogs were due to species, to some unrecognized difference between
    dieldrin and aldrin, to the fact that the rats received lower dosages,
    or to some unidentified factor.

        Studies of the distribution of DDT in various lipid fractions that
    are based on tissue extracts obtained with one or more organic
    solvents, such as those of Kuz'minskaja et al. (1972), are difficult
    to interpret because there is no way of determining how much of the
    material was initially associated with protein.

    DDE

        DDE constitutes about 4% of technical DDT. Most species convert
    some of the DDT they ingest to DDE. Finally, most species, including
    man, store DDE more tenaciously than they do DDT, the greater part of
    which is metabolized by a different pathway to DDA and excreted more
    rapidly. The result is that DDE, expressed as a percentage of total
    DDT-related compounds, increases in individuals after DDT intake
    decreases, and also increases in successive steps of the food chain.

        Apparently, the rhesus monkey is an exception. Monkeys store DDE
    when it is fed to them but, when feeding stops, the rate of loss of
    DDE stored in fat is more rapid than that of DDT (Durham et al.,
    1963). Whether it is relative inability to form DDE, unusual ability
    to excrete it, or a combination of both that accounts for the fact
    that little or no DDE can be found in monkeys fed DDT is not entirely
    clear.

    6.3  Elimination

    6.3.1  Human studies

    6.3.1.1  Studies of volunteers

        DDA is the main urinary metabolite of DDT. In man, it was found
    first in a volunteer by Neal et al. (1946), who reported that,
    following ingestion of 770 mg of  p,p'-DDT, excretion rose sharply to
    4.0 mg/day during the second 24-h period, decreased rapidly on the
    third and fourth days, decreased gradually thereafter, but was still
    above baseline on the fourteenth day. Judging from a graph, the
    highest concentration was about 2.6 mg/kg.

        Much later studies in volunteers, who received smaller but
    repeated doses, showed that a steady state of excretion was reached
    after about 6-8 months. During a 56-week period of continued dosing
    after equilibrium was fully established, the concentration of DDA
    associated with technical DDT at the rate of 35 mg/man per day varied
    from 0.18 to 9.21 mg/kg and averaged 2.98 mg/kg, corresponding values
    for  p,p'-DDT were 0.40-6.27 mg/kg with a mean of 1.88 mg/kg. Thus,
    technical DDT was excreted more effectively and stored in man less
    than  p,p'-DDT. During the latter half of the dosing period, it was
    possible, in the 2 groups receiving recrystallized and technical DDT
    at the rate of 35 mg/man per day, to account for an average of 13% and
    16%, respectively, of the daily dose in terms of urinary DDA. The
    excretion of DDA was relatively constant in each individual, but
    marked differences were observed between men receiving the same dose.
    For example, over the period of 56 weeks, the highest rate measured
    for one man was 0.16 mg/h while the lowest rate for another in the
    same group was 0.15 mg/h. Their mean rates during this period were
    0.089 and 0.269 mg/h, respectively. The difference was highly
    significant (P < 0.001) (Hayes et al., 1971).

    6.3.1.2  Studies of occupationally exposed workers

        Among workers whose DDT intake was estimated to be about 35 mg/man
    per day, Ortelee (1958) reported that the concentration of DDA in
    urine ranged from 0.12 to 7.56 mg/litre and averaged 1.71 mg/litre.
    Among workers whose exposure was about half as high, Laws et al.
    (1967) found concentrations from 0.01 to 2.67 mg/litre with a mean of
    0.97 mg/litre.

        Continuous sampling of a DDT-formulating plant worker's urine
    showed that excretion of DDA increased promptly, when exposure began
    on each of 5 consecutive work days and often continued to increase
    after exposure, sometimes reaching a peak about midnight before
    decreasing rapidly. On the sixth day, when there was no occupational
    exposure to DDT, the excretion of DDA continued until a very low level
    was reached. The highest concentration of DDA reported in this study
    was 0.68 mg/litre (Wolfe & Armstrong, 1971).

    6.3.1.3  Studies of the general population

         Urine. Cranmer et al. (1969) developed a method for analysing
    DDA in urine that, for the first time, made it possible to measure the
    compound in every sample. The range found for a small sample of the
    population of Florida, USA, was 0.008-0.019 mg/litre, and the mean
    (0.014 mg/litre was slightly less than 0.02 mg/litre, the lowest
    concentration detectable by earlier methods. Results for general
    population studies are shown in Table 14 together with results for
    various groups of workers and volunteers.

        Table 14.  Urinary excretion of DDA by people in the USA with various degrees of exposure to DDTa

    Exposure                 Year       No. of     DDA excretion (mg/kg)      Reference
                                        samples                                                   
                                                   Range             Mean

    General population       1954          8      <0.05              --       Hayes et al., 1956
    General population       1957          8      <0.02-0.07         --       Hayes et al., 1971
    General population       1962         23      <0.02-0.18         --       Durham et al., 1965b
    General population       1968         11       0.008-0.019       0.014    Cranmer et al., 1969
    Environmentalb           1962         13      <0.02-0.11         --       Durham et al., 1965a
    Subjects applying DDT    1962         11      <0.02-0.17         --       Durham et al., 1965a
    Formulators              1957         40       0.12-7.56         1.71     Ortelee, 1958
    Makers and formulators   1966         35      <0.01-2.67         0.97     Laws et al., 1967
    Volunteers given         1953-1954     2       0.10-0.42b        0.21c    Hayes et al., 1956
      3.5 mg/day orally
    Volunteers given         1957-1958     6       0.06-1.98a        0.23b    Hayes et al., 1971
     3.5 mg/day orally
    Volunteers given         1953-1954     6       0.69-9.67b        2.46c    Hayes et al., 1956
      35 mg/day orally
    Volunteers given         1957-1958     6       0.18-9.21c        3.09d    Hayes et al., 1971
      35 mg/day orally

    a  Slightly modified from Hayes (1975).
    b  Residents living within 500 feet of agricultural application.
    c  Based on all samples after thirty-fifth week of dosage.
    d  Based on all samples from the thirty-fifth to the ninety-third week after dosage started.

        In the general population the urine contained not only DDA but
    also neutral compounds; the average concentrations reported by Cueto &
    Biros (1967) were:  p,p'-DDT, 0.0007 mg/litre and  p,p'-DDE,
    0.0156 mg/litre. Men with full-time occupational exposure to DDT
    excreted much more DDA but showed only a statistically insignificant
    increase in the excretion of DDT and DDE.

        These values for the excretion of DDA, DDT, and DDE by different,
    small groups of people showed an average concentration of
    0.0358 mg/litre of DDT-related material. Although the DDT intakes of
    these particular groups were not measured, the urinary excretion is of
    such an order of magnitude that it may account for the excretion of
    all the absorbed DDT.

         Milk. As far as the mother is concerned, the secretion of a
    compound in milk is a form of excretion. For the infant, the milk is
    an important, if not the sole source of intake of the compound in
    question. The concentrations of DDT and DDE in milk reported from
    different countries are listed in Table 15. Especially high values
    have been reported from Guatemala for 1970 (see Table 16) and from the
    USSR (Damaskin, 1965). However, additional and more numerous samples
    taken only four years later in the same and other communities in
    Guatemala revealed entirely different results. The highest single
    value observed for total DDT in 1974 was 5.69 mg/litre. The range of
    average values for different locations was 0.04-0.86 mg/litre. The
    means for total DDT for the three communities studied earlier were: La
    Bomba, 0.59 mg/litre; El Rosario, 0.28 mg/litre; and Cerro Colorado,
    0.47 mg/litre (Winter et al., 1976). The authors recognized the
    importance of the agricultural uses of DDT as a potential source of
    the compound in human milk. However, they attributed the change
    between 1970 and 1974, almost exclusively, to the substitution of
    propoxur for DDT in residential spraying to combat malaria.

        The medical importance of DDT in human milk depends entirely on
    the dosage of the compound received by babies. The highest
    concentration of  p,p'-DDT ever reported in a single sample of milk
    and the highest average value from one community (see Table 16) would
    determine maximum and average intakes of 1.06 and 0.32 (mg/kg) day for
    newborn babies, assuming an intake of 0.6 litre per day and a weight
    of 3.36 kg. This average intake is of the same order of magnitude as
    that encountered by workers who make and formulate DDT. The
    corresponding maximum and average intakes of total DDT-related
    material for infants in Guatemala would be 2.18 and 0.73 (mg/kg) day,
    values not strictly comparable to those of the workers because the
    intake of the babies includes so much more DDE, a less toxic compound.
    Neither of the papers cited concerning DDT in human milk in Guatemala
    mentioned any indication of injury to babies. Absence of injury to
    babies would be predicted from studies of the most heavily exposed
    workers and also from studies regarding the effect of age on the
    susceptibility of animals to DDT (see section 7.3.2).

        Table 15.  Concentration of DDT-derived material in human milk

    Country            Year        No. of    Method     DDT         DDE as DDT   Total as      DDE as      Reference
                                   samples   of         (mg/litre)  (mg/litre)   (mg/litre)    DDT
                                             analysis                                          (% of
                                                                                               total)

     North America
    Canada             1967-1968     147      GLC       0.032         0.097       0.139         --      Ritcey et al., 1972
    Canada                            15      GLC       0.006-0.032   --          0.019-0.035   --      Musial et al., 1974
    USA                1950           32      colour                  --          0.13          --      Laug et al., 1951
    USA                1960-1961      10      colour    0.08          0.04        0.12          33      Quinby et al., 1965b
    USA                1962            6      colour    0-0.12a       0.025a      0.37b         --      West, 1964
    USA                1968            ?      GLC       0.026         0.047       0.078         60      Cudey & Kimbrough, 1969
    USA                1970           53      GLC       0.022         0.083       0.101         80      Kroger, 1972
    USA                1970-1971     101      GLC       --            --          0.17                  Wilson et al., 1973
    USA                1971-1972      40      GLC                                 0.126                 Savage et al., 1973
    USA                1973-1974      57      GLC       0.092         0.260       0.344         76      Strassmann & Kutz, 1977
    USA                1974           38      GLC                                 0.447                 Woodard et al., 1976
    USA                1974           14      GLC                                 0.075                 Woodard et al., 1976
    USA                1975            7      GLC                                 0.323                 Woodard et al., 1976

     Europe
    Belgium            1968           20      GLC       0.05          --          --            --      Heyndrickx & Maes, 1969
    Czechoslovakia     1968           --      --        0.101         --          --            --      Hruska, 1969
    Czechoslovakia                   393      TLC       0.097         0.112       0.209         54      Suvak, 1970
    England            1963-1964      19      GLC       0.05          0.08        0.13          62      Egan et al., 1965
    France             1971-1972                                                  3.24c                 Luquet et al., 1974
    France             1972?                                                      3.24c                 Luquet et al., 1974
    German Democratic  1970?                                                      0.569                 Adamovic et al., 1971
    Republic
    German Democratic  1969           57                                          0.23                  Engst & Knoll, 1972
    Republic
    German Democratic  1970           18                                          0.16                  Engst & Knoll, 1972
    Republic

    Table 15 (Cont'd)

    Country            Year        No. of    Method     DDT         DDE as DDT   Total as      DDE as      Reference
                                   samples   of         (mg/litre)  (mg/litre)   (mg/litre)    DDT
                                             analysis                                          (% of
                                                                                               total)

    German Democratic  1971           96                                          0.32                  Knoll & Jayarman, 1973a
    Republic                                                                                            1973b
    Germany, Federal   1970?          43      GLC       0.031         0.090       0.121         74      Acker & Schulte, 1970
    Republic of
    Germany, Federal   1971?                                                      0.121                 Pfeilsticker, 1973
    Republic of
    Hungary            1963           10      colour    0.13-0.26a    --          --            --      Denés, 1964
    Italy              1965?           2      GLC       0.001         0.055       0.056                 Kanitz & Castello, 1966
    Netherlands        1969           50      GLC       0.9c          1.8c        2.7c          66      Tuinstra, 1971
    Poland             1966           26      colour    0.27          --          --            62      Bronisz & Ochynski, 1968
    Poland             1967           25      colour    0.40          --          --            58      Bronisz & Ochynski, 1968
    Poland             1970?          40      GLC       0.08          0.19        0.28          71      Kontek et al., 1971
    Portugal           1972          168      GLC                                 0.326                 Graca et al., 1975
    Romania            1968?         100      colour    0.054-0.749   0.026-8.30  0.080-9.05    --      Unterman & Sirghie, 1969
    Sweden             1967?          --                --                        0.117         --      Lotroth, 1968
    Sweden             1967-1969      22      GLC       0.039         0.076       0.115         63      Westoo et al., 1970
    USSR               1964           16      colour    1.22-4.88     --          --            --      Damaskin, 1968
    USSR               1968?        4505      --        0.1-1.0       --          --            --      Gracheva, 1969
    USSR               1969?          --      --        0.09          --          0.14          --      Gracheva, 1970
    USSR               1967?         370      GLC       0.1           --          --                    Komarova, 1970

     Asia
    Israel             1975           29      GLC       0.02          0.03        0.07          44      Polishuk et al., 1977
    Japan              1970?          10                                          0.071                 Tokutsu et al., 1970
    Japan              1970?           5                                          0.160                 Takeshita & Inuyama. 1970
    Japan              1970?          10                                          0.120                 Takeshita & Inuyama. 1970
    Japan              1971?                                                      0.04                  Kojima et al., 1971
    Japan              1971?                                                      0.04                  Kojima et al., 1971

    Table 15 (Cont'd)

    Country            Year        No. of    Method     DDT         DDE as DDT   Total as      DDE as      Reference
                                   samples   of         (mg/litre)  (mg/litre)   (mg/litre)    DDT
                                             analysis                                          (% of
                                                                                               total)

    Japan              1971?          59                                          0.019-0.105           Kato et al., 1971
    Japan              1971?          14                                          0.047                 Sugaya et al., 1971
    Japan              1971           43      GLC       0.095         0.084       0.179         47      Hidaka et al., 1972
    Japan              1971          454      GLC                                 0.0607                Hayashi, 1972a, 1972b
    Japan              1971-1972     398                                          0.0626                Hayashi, 1972a, 1972b
    Japan              1971-1972     398                0.0562        --          --            --      Anonymous, 1972
    Japan              1971                                                       0.044                 Yamagishi et al., 1972
    Japan                             30                                          2.0a                  Mizoguchi et al., 1972
    Japan                             54                                          0.035                 Taira et al., 1972
    Japan              1971-1972       5                                          0.027                 Nagai, 1972
    Japan              1971-1972       5                                          0.037                 Nagai, 1972
    Japan              1971-1972       5                                          0.016                 Nagai, 1972
    Japan              1971-1972       5                                          0.037                 Nagal, 1972
    Japan                             30                                          0.033                 Oura et al., 1972
    Japan              1971-1972     123                                          0.105                 Kawai et al., 1973
    Japan              1971-1972                                                  0.038-0.075           Kamata, 1973
    Japan              1970                                                       3.780c                Suzuki et al., 1973
    Japan              1971                                                       3.592c                Suzuki et al., 1973
    Japan              1972                                                       3.822c                Suzuki et al., 1973
    Japan              1973                                                       0.0854                Kamata, 1974

     Oceania
    Australia          1970            1      GLC       --            --          0.014d                Newton & Greene, 1972
                                                                                  0.007e
                                                                                  0.066f
                                      67      GLC       0.036         0.105       0.141

    Table 15 (Cont'd)

    Country            Year        No. of    Method     DDT         DDE as DDT   Total as      DDE as      Reference
                                   samples   of         (mg/litre)  (mg/litre)   (mg/litre)    DDT
                                             analysis                                          (% of
                                                                                               total)
                                                                                                                                
    Australia
    (Brisbane)         1971-1972      20      GLC                                 0.288                 Miller & Fox, 1973
    (Mareeba)          1971-1972      20      GLC                                 0.415
    Australia                         45      GLC                                 0.064                 Siyali, 1973
    Australia                         22      GLC       0.010         0.068       0.078                 Stacy & Thomas, 1975
    Papua New Guinea   1972           16      GLC
    (Kar Kar Island)                                    0.002         0.002       0.004         50      Hornabrook et al., 1972
    Papua New Guinea   1972           19      GLC
    (Sepik district)                                    0.008         0.007       0.015         47      Hornabrook et al., 1972

    a  Range of values for milk containing 4% fat    c Concentration in milkfat.             e At middle of feeding, 1.2% fat.
       containing 3.3-6.6 ppm.
    b  Maximal value.                                d At beginning of feeding, 1.8% fat.    f At end of feeding, 5.1% fat.

        Table 16.  Ranges, means, and standard errors of the concentrations
               of organochlorine insecticides in the milk of women in
               three towns in Guatemalaa

    Compound              La Bomba          El Rosario          Cerro Colorado
                          1970              1970                1971
                          n = 10            n = 27              n = 9

    p.p'-DDT              0.23-4,95         0.16-2.24           0.49-5.94
    (mg/litre)            (1.00 ± 0.38)     (0.77 ± 0.10)       (1.78 ± 0.56)
    p.p'-DDE              0.12-6.36         0.28-3,10           0.60-6.13
    (mg/litre)            (1.02 ± 0.58)     (0.99 ± 0.14)       (2.10 ± 0.61)
    p,p'-TDE(DDD)         trb-0.16          0.01-0.09           0.05-0.11
    (mg/litre)            (0.03 ± 0.02)     (0.02 ± 0.004)      (0.07 ± 0.01)
    o,p'-DDT              tr-0.29           0.01-0.18           0.06-0.22
    (mg/litre)            (0.09 ± 0.03)     (0.06 ± 0.01)       (0.12 ± 0.02)
    total as DDT          0.41-11.50        0.34-4.97           1.571-12.21
    (mg/litre)            (2.15 ± 1.05)     (1.84 ± 0.24)       (4.07 ± 1.11)
    total HCH             0.01-0.10         tr-0.07             0-0.06
    (mg/litre)            (0.03 ± 0.01)     (0.007 ± 0.003)     (0.02 ± 0.01)
    heptachlor epoxide    0-0.02            tr-0.01             tr
    (mg/litre)            (0.003 ± 0.002)   (0.007 ± 0.0004)
    dieldrin              tr                tr-0.01
    (mg/litre)                              (0.002 ± 0.0005)

    a  From: Olszyna-Marzys et al. (1973).
    b  tr = trace.

        The slightly greater secretion of DDT and much greater secretion
    of various isomers of HCH by urban mothers (compared to rural mothers)
    in Japan was attributed to their greater intake of cow's milk (Takano,
    1972).

         Other routes of excretion. DDT, DDE, and dieldrin are excreted in
    the bile; the concentrations for five men without special exposure
    varied as follows:  p,p'- and  o,p'-DDT combined, 0.0000-0.0009 mg/litre;
     p,p'-DDE, 0.0005-0.0056 mg/litre; and dieldrin, 0.0000-0.0005 mg/litre.
    Higher levels were found in the bile of one pest control operator
    (Paschal et al., 1974).

    6.3.2  Animal studies

        When large doses of DDT are ingested, some of the compound is not
    absorbed and is passed in an unaltered state in the faeces. Only
    traces of unaltered DDT may be found in the faeces when exposure is by
    any route other than oral. However, true faecal excretion of DDT
    metabolites was established very early (Wasicky & Unti, 1945; Judah,
    1949). In the rat, faecal excretion of DDT exceeded urinary excretion,
    irrespective of the route of administration (Hayes, 1965). In man, the
    ratio is obviously different. Although the excretion of DDT-related
    material in the faeces of man receiving 35 mg/man per day has been
    reported using colorimetry (Hayes et al., 1956), this result has never
    been confirmed by gas chromatography, even in connexion with workers
    whose exposure was heavy and prolonged. Either DDT metabolites are not
    excreted by man in the faeces to any great degree, or they are
    excreted in one or more forms that differ from those already
    demonstrated in rats.

        The bile appears to be the principal source of DDT metabolites in
    the faeces of rats. When the bile duct was cannulated before
    intravenous injection of radioactive DDT, 65% of the dose was
    recovered in the bile, 2% in the urine, and only 0.3% in the faeces
    (Jensen et al., 1957), and the possibility of some contamination of
    the faeces by urine could not be excluded.

        The different routes of excretion are not unrelated. Burns et al.
    (1957) found that there was an increase in urinary excretion of
    radioactive material following ligation of the bile duct in rats fed
    radioactive DDT. This is an indirect confirmation of the finding by
    Jensen and his colleagues that most of the metabolites in bile consist
    of DDA or compounds closely related to it. Although an enterohepatic
    circulation of the metabolites of DDT has not been directly proved, it
    seems likely that such a circulation exists, as has been demonstrated
    for 1,1'-(2,2-dichloroethylidene)-bis[4-ethylbenzene] (Perthane).

        Demonstration of the excretion of DDT in milk was first reported
    by Woodard et al. (1945) in connexion with a dog fed the compound at
    the rate of 80 (mg/kg) day. Within a short time, excretion of DDT in
    milk was reported in rats, goats, and cows, and soon afterwards, in
    women (Laug et al., 1951). Telford & Guthrie (1945) showed that rats
    fed a diet containing DDT at 1000 mg/kg produced milk that was toxic
    to their young.

        Since the early laboratory studies, the presence of DDT has been
    demonstrated repeatedly in the milk of cows. A review (Hayes, 1959)
    showed that cows fed substantial, but nontoxic, residues of DDT
    commonly excrete 10% or slightly more of the total dose in their milk,
    and amounts slightly over 30% have been observed.

        Further information on the excretion of DDT in human milk is given
    in section 6.3.1.3. It is of interest to repeat here, however, that
    lactating women in the general population are apparently in negative
    DDT balance. That is, they excrete more DDT in their milk each day
    than they acquire in their food. The difference is small and would not
    be expected to have much effect on their total body burden of DDT.

        Wilson et al. (1946a) showed that DDT was secreted from the skin
    of a cow maintained on an oral dosage of about 53 (mg/kg) day.

         DDA. Because DDA is the main form in which DDT is excreted, it
    might be expected that, following its direct administration, DDA would
    be excreted relatively efficiently, and this is true. It was found
    very early that, during the first few days after oral dosing, rabbits
    excreted DDA in the urine approximately 15 times faster than animals
    given DDT at an equivalent dosage. Although the rate of DDA excretion
    increased somewhat, the rate of excretion associated with DDT
    increased more rapidly so that the values differed by a factor of only
    five after the twentieth day of feeding (Smith et al., 1946).

    6.4  Biotransformation

        The chemical nature of the chief metabolite excreted in the urine
    was first elucidated by White & Sweeney (1945). Rabbits were given DDT
    (melting point 107-108°C) at a rate of 100 (mg/kg) day for 6 days per
    week, and their urine was collected. It contained a considerable
    amount of organic chloride, whereas normal rabbit urine did not. Using
    the organic chloride test to evaluate different methods of extraction,
    the authors were able to isolate a crystalline material containing
    25.37% chlorine and melting at 166-166.5°C. The crystals were shown to
    be DDA (see Table 1). The product obtained from the urine was
    identical to that synthesized from glyoxylic acid and chlorobenzene
    and with a compound obtained through the chemical degradation of DDT.
    Identity of the 3 compounds and, therefore, their true chemical
    nature, was established by the determination of melting points, mixed
    melting points, elementary analysis, and X-ray powder diffraction
    patterns, as well as by demonstrating the similarity of the
    decarboxylation products of the three original materials. Only 80-85%
    of the total organic chloride of the rabbit urine was found to be
    soluble in alkali and in bicarbonate. For this and other reasons it
    was considered possible that DDA was not the only chlorinated organic
    compound present.

        Later work by many authors has confirmed that DDA is the major
    urinary metabolite of DDT in all mammals including man. It may be
    added that, in spite of great strides in analytical chemistry, the
    nature of other urinary metabolites has not been elucidated fully.

        The fact that DDE is stored in tissue was first demonstrated by
    Pearce et al. (1952) in connexion with human fat. The authors pointed
    out that they did not know whether the compound resulted from partial

    degradation of DDT residues on plants or whether the DDE was formed
    during the process of digestion or after absorption. It is now known
    that some food contains DDE but that man is capable of forming the
    product from DDT. The exact mechanism of the biotransformation of DDT
    to DDE remains in doubt.

        Pearce et al. (1952) established the identity of DDE by comparing
    the colorimetric and column chromatographic behaviour of the residue
    with those of a chemical standard. A second paper from the same
    laboratory (Mattson et al., 1953) added further details confirming the
    identity of the compound. Later investigations have confirmed the
    identity of DDE by infrared spectrometry and by gas chromatography.

        That portion of the metabolism of DDT that leads to DDA has been
    clearly elucidated by Peterson & Robinson (1964), who gave evidence
    for the sequence of changes shown in Fig. 2. Organ perfusion studies
    have indicated that the liver is capable of the biotransformation of
    DDT, DDE, TDE, DDMU, and DDMS, while the kidney transforms DDMS, DDNU,
    and DDOH (Datta & Nelson, 1970). Cultures of embryonic lung cells are
    capable of metabolizing DDT to DDA via DDD (North & Menzer, 1973).

        When DDA was discovered, it was postulated, on chemical grounds,
    that DDE was a step in its formation (White & Sweeney, 1945); however,
    rats that produced both DDE and DDA from DDT were incapable, according
    to Peterson & Robinson (1964), of forming DDA when fed preformed DDE.
    This finding was contradicted by Datta (1970) and by Datta & Nelson
    (1970) who claimed that 14C- p,p'-DDE was converted by rats to
     p,p'-DDMU, which then underwent further metabolism to  p,p'-DDA via
    the route shown in Fig. 2. Datta suggested that the predominance of
    detoxication via DDE or TDE (DDD) might depend on physiological
    response or the amount of toxicant used. Whatever the reason, the fact
    remains that DDE is stored more tenaciously than DDT.

        Rhesus monkeys fed either technical or  p,p'-DDT store little or
    no DDE, although they are fully capable of storing DDE when it is fed
    preformed (Durham et al., 1963).

        The way in which DDE was lost from storage was not clearly
    understood for a long time. In man (Cueto & Biros, 1967), seal, and
    guillemot (Jansson et al., 1975) part of it is excreted unchanged, but
    the fact that its elimination is promoted by the induction of
    microsomal enzymes (see section 7.1.4.2) strongly suggests that it
    undergoes metabolism, conjugation, or both. That metabolism does occur
    was first demonstrated by identification of 2 hydroxylated derivatives
    of DDE in the faeces of wild seals and guillemots and in the bile of
    seals (Jansson et al., 1975). When  p,p'-DDE was fed to rats, the
    same metabolites and one other were isolated from the faeces, and,
    within the first 6 days, the metabolites accounted for about 5% of the
    dose (Sundström et al., 1975). Later a fourth hydroxylated derivative
    was identified from the faeces of rats fed  p,p'-DDE. The compounds

    are  m-hydroxy- p,p'-DDE [1,1-dichloro-2-( p-chloro- m-hydroxyphenyl)-
    2-( p-chlorophenyl)-ethylene, the major metabolite],  o-hydroxy- p,p'-DDE,
     p-hydroxy- m,p'-DDE (the product of an NIH shift), and  p-hydroxy-
     p'-DDE. A scheme (Fig. 3) involving  m,p-epoxy- p,p'-DDE and  o,m-epoxy-
     p,p'-DDE was proposed for the formation of these metabolites as
    well as a fifth metabolite (Sundström, 1977). Neither the fifth
    metabolite nor the hypothetical intermediate have been isolated.

        DDE is metabolized not only to easily excretable phenols but also
    to  m-methylsulfone- p,p'-DDE. In the blubber of seals from the
    Baltic, this compound was found in a concentration of 4 mg/kg along
    with DDE (138 mg/kg), TDE (DDD) (10 mg/kg), DDT (78 mg/kg) and various
    PCB's and their metabolites (150 mg/kg) (Jensen & Jansson, 1976).

        The conversion of  o,p'-DDT to  p,p'-DDT has been reported
    (Klein et al., 1965; French & Jefferies, 1969), but, when the
    possibility was reinvestigated using 14C- o,p'-DDT, no conversion
    could be detected (Cranmer, 1972). The chromatographic peak closely
    resembling that of  p,p'-DDT observed in the earlier studies
    undoubtedly was due to the presence of a metabolite of  o,p'-DDT,
    which may explain the more rapid metabolism of the  o,p'-isomers that
    has been observed in rat, man, and perhaps other species. The more
    rapid excretion of  o,p'-DDT is explained, at least in part, by the
    observed ring-hydroxylation of the parent compound in rats (Feil et
    al., 1973) and chickens (Feil et al., 1975) and of preformed  o,p'-TDE
    (DDD) in rats (Reif & Sinsheimer, 1975) and in man (Reif et al.,
    1974). At least 13 metabolites were detected in rats and 15 in
    chickens. Ring-hydroxylation, which has not been observed with  p,p'-
    DDT or  p,p'-TDE, was present in all species. There were, however,
    some species differences. For example,  o,p'-DDE and three
    hydroxylated  o,p'-DDE's were found in the excreta of chickens but
    not in the excreta of rats. In 2 patients with adrenal carcinoma for
    which they were receiving  o,p'-TDE at a rate of 2000 mg/day, as much
    as 46-56% of the daily intake was recovered in the urine following
    acid hydrolysis. Just over half of the recovered material was in the
    form of  o,p'-DDA, but the remainder was in the form of hydroxylated
    derivatives, specifically  m-hydroxy,  p-hydroxy-,  m-hydroxy- p-
    methoxy-, and  p-hydroxy- m-methoxy- o,p'-DDA. Some other
    hydroxylated compounds were found in trace amounts. All hydroxylation
    had occurred on the ring that had its chlorine in the ortho position
    (Reif et al., 1974). When the metabolism of a single 100 mg oral dose
    of 14C- o,p'-TDE was studied in rats, averages of 7.1 and 87.8% of
    the activity were recovered in the urine and faeces, respectively,
    within 8 days (Reif & Sinsheimer, 1975). The high recovery indicated
    rapid excretion with little storage.

        The compound identified by Peterson & Robinson (1964) as a
    "probable intermediate aldehyde" was later synthesized and shown to be
    highly labile (McKinney et al., 1969), confirming the guess by
    Peterson & Robinson that it was unlikely to accumulate in tissues in
    measurable amounts.

        Of the compounds shown in Fig. 2 and 3, only DDT, TDE (DDD), DDE,
    and DDA are commonly reported in the tissues or excreta of animals,
    including man. The symptomatology produced when the metabolites are
    administered directly is discussed in section 7.1.1, while uptake,
    distribution, and elimination of the compounds are discussed in
    sections 6.1.2, 6.2.2, and 6.3.2, respectively.

        Although microorganisms, plants, insects, and birds produce many
    of the same metabolites that are found in mammals, there are
    interesting differences. Nearly 20 derivatives (including mammalian
    metabolites) have been identified, and the chemical structure of
    several more is still unknown. Some aspects of nonmammalian, as well
    as mammalian metabolism have been reviewed (Menzie, 1969; Klein &
    Korte, 1970; Fishbein, 1974; Schroeder & Dorozalska, 1975). The
    metabolism of microorganisms and plants, as well as that of domestic
    animals, may influence the composition of DDT-derived residues in
    human food, but there is no evidence that these residues contain a
    significant amount of any compound not formed from DDT by human
    metabolism.

    7.  EXPERIMENTAL STUDIES ON THE EFFECTS OF DDT

    7.1  Animal Studies

    7.1.1  Haemopoietic system and immunology

        Many early reports reviewed by Hayes (1959) indicated that large
    doses of DDT might not have any effect on the blood or that they might
    produce a moderate leukocytosis and a decrease in haemoglobin, with or
    without a decrease in the concentration of red cells. The leukocytosis
    probably is secondary to stimulation of the sympathetic nervous
    system, while the loss of haemoglobin may be nutritional in origin.
    Later studies have confirmed the early results. A range of
    haematoiogical variables remained unchanged in squirrel monkeys dosed
    orally at rates of 0, 0.05, 0.5, 5, and 50 (mg/kg)/day, even though
    the highest dosage was fatal within 14 weeks (Cranmer et al., 1972).

         Immunology. Inasmuch as some compounds are antibiotic, it is
    logical that some may be probiotic, that is, that they either reduce
    resistance to infection or increase the virulence of an infecting
    organism (Hayes, 1975). Far fewer studies have been made of probiosis
    than of antibiosis. A number of papers have reported one or other
    possibly probiotic property of DDT, but some of the reports could not
    be confirmed, and others have not been retested.

        It has been claimed that a change in the phagocytic activity of
    white blood cells is an indication of early intoxication by DDT
    (Kun'ev, 1965). However, Kaliser (1968) did not find any statistical
    difference in  in vitro or  in vivo phagocytosis of control rats and
    those receiving DDT by stomach tube at a rate of 0.25 (mg/kg)/day for
    31 days. The highest rate at which men who make and formulate DDT in
    the USA now absorb the compound is about 0.25 (mg/kg)/day.

        Rats receiving an aqueous suspension of  p,p'-DDT of unstated
    stability at a concentration of 200 mg/litre as their only source of
    water for over 30 days were reported to develop a lower titre of
    antibodies to ovalbumen (Wassermann et al., 1969). Rabbits responded
    to the same treatment with a statistically significant reduction of
    antibody titre against  Salmonella and a reduction in antibody titre
    against sheep red blood cells that was not statistically significant
    (Wassermann et al., 1971). Both the rats and rabbits showed a decrease
    in at least one globulin fraction of the blood.

        Other reports of changes in immunohaematological indices are those
    of Semenceva (1968) and Fridman (1970).

        One group of investigators has shown clearly that what at first
    appeared to be an immunological response really involved a quite
    different, predictable effect. Briefly, it was shown that guineapigs
    sensitized to diphtheria toxoid were less susceptible to anaphylaxis

    in response to a challenge dose of the toxoid if they were pretreated
    with DDT at a dosage of only 1-20 mg/kg. Direct measurement of
    antitoxin production indicated little or no difference between
    protected and unprotected animals. Furthermore, some protection was
    given by DDT administered for only 3 days prior to the induction of
    anaphylaxis (Gabliks et al., 1973, 1975). Further studies showed that
    DDT treatment reduced the histamine levels in the lungs of both
    immunized and nonimmunized animals. The number of detectable mast
    cells was also reduced; this was true whether the count was made in
    tissues from guineapigs dosed systemically with DDT or in the lungs
    and mesenteric tissue taken from untreated animals and exposed to DDT
     in vitro at concentrations ranging from 10 to 45 mg/litre. These
    results indicate that the protection offered by DDT was the result of
    a reduction of the amount of histamine available for sudden release in
    response to a challenge dose of toxoid (Askari & Gabliks, 1973).
    Regardless of exposure to DDT, immunization leads to an increase in
    detectable mast cells (Gabliks et al., 1975).

    7.1.2  Nervous system and behaviour

        DDT intoxication in animals was well described by Domenjoz (1944).
    The first perceptible effect is abnormal susceptibility to fear, with
    violent reaction to normally subthreshold stimuli. There is definite
    motor unrest and increased frequency of spontaneous movements. As
    poisoning increases, hyperirritability, like that seen in strychnine
    poisoning develops, but convulsions do not appear at this time. A fine
    tremor, recognizable at first only as a terror reaction, is later
    present as all intention tremor in connexion with voluntary movement.
    Then it is present intermittently without observable cause, and
    finally it is present as a coarse tremor without interruption for as
    long as several days. Spontaneous movement is limited, and food intake
    stops so that surviving animals lose weight. In the later stages,
    especially in some species, there are attacks of epileptiform, tonic-
    clonic convulsions with opisthotonos.

        All the signs are strengthened by external stimuli and become
    manifest at first through external stimuli. At all stages, the animals
    show normal position and labyrinth reflexes. The picture of poisoning
    in mammals recalls the disturbances of movement and tone that are
    known in human pathology as the amyostatic syndrome.

        Symptoms appear several hours after oral administration of the
    compound, and death follows after 24-72 h. The latent period after
    intravenous administration at about the LD50 level is approximately
    5 min; signs of poisoning reach a maximum level in about 30 min, and
    survivors are symptom-free in 18 to 24 h. Animals that survive recover
    completely.

        In addition to the features of poisoning already mentioned,
    Cameron & Burgess (1945) noticed that as rats, guineapigs, and rabbits
    become sick, they become cold to the touch and show ruffled fur. Some

    show diarrhoea. These authors found that muscular tremors were
    preceded by muscular weakness that first occurred in the back and
    later in the hind legs. The front legs were relatively spared so that
    animals showing marked weakness of the hind quarters could still drag
    themselves about. However, several authors have found that the tremor
    characteristic of DDT poisoning generally starts in the muscles of the
    face, including the eyelids, and spreads caudally with variable
    severity until all the muscles are affected. Furthermore, although
    weakness of hind quarters has been seen by others, it is not a common
    finding.

        Like tremor, coldness of the skin and ruffling of the fur probably
    represent an indication of disturbed thermal regulation. Apparently it
    was not until the work of Hrdina et al. (1975) that an increase of
    almost 3°C in body temperature was reported in rats following a fatal
    (600 mg/kg) oral dosage of DDT.

        Although there is a general similarity in the clinical effects of
    DDT in all vertebrate species, some characteristic differences exist.
    Cats show greater extensor rigidity and opisthotonos than other
    laboratory animals. The stiffness appears first in the distal part of
    the extremities and later extends to the proximal part and to the
    trunk. Poisoned cats show marked pilomotor activity. Convulsions in
    them may become almost continuous. Convulsions are also prominent in
    dogs as is ataxia. Tremors are so pronounced in rats that it may be
    difficult to detect clonic convulsions in them. Rats poisoned by DDT
    show a reddish colour about the eyes just as they do when ill from
    many other causes. The colour has been attributed to excessive
    secretion of a porphyrin by the Harderian glands.

        Poisoning produced by repeated doses of DDT differs from that
    produced by a single dose only in so far as the animal may be
    gradually debilitated, especially by malnutrition. If food intake is
    maintained, tremor may last for weeks, or even intermittently, for
    months. If the animals survive a short time after dosing stops,
    recovery is complete. However, food intake may be interfered with in
    at least two ways. Tremor and more severe signs may interfere
    mechanically with eating. Animals offered food containing high
    concentrations of DDT often eat little or nothing and lose weight
    rapidly. However, the same animals will show excellent appetites when
    offered the same kind of food without DDT just after refusing the
    major portion of the daily ration of contaminated food. Unlike
    dieldrin and some other compounds, DDT seems to have little effect on
    appetite as mediated by the central nervous system; it has a great
    deal to do with taste.

        Animals that have suffered severe weight loss as a result of DDT
    poisoning may die partly as a result of general debility. In some
    colonies, at least, they have become prey to secondary infection.

        In summary, it may be said that animals that die as the result of
    repeated large doses of DDT and small animals that die as a
    complication of starvation following many somewhat smaller doses of
    DDT show the same signs as those seen in animals killed by one or a
    few large doses. Even though severely ill, animals that survive a few
    days after the last of many doses of DDT recover.

        Of samples that may be collected from a living animal, the
    concentration of DDT in serum most accurately reflects its
    concentration in the brain, the critical tissue. In the rat, levels of
    25 mg/kg (wet weight) in the brain are not usually fatal: higher
    levels tend to be fatal regardless of whether absorption followed one
    or many doses (Dale et al., 1963; Hayes & Dale, 1964). As reviewed by
    Hayes (1975), the danger level is approximately the same in several
    species of birds.

        Behavioural changes may be demonstrated in animals receiving DDT,
    daily, at rates too low to produce illness. Khairy (1959) was able to
    detect ataxia in the form of changes in gait in rats that had been fed
    DDT at dietary levels of 100 mg/kg or more for 21 or more days. Gait
    was recorded by smearing the hind paws of the animals with vaseline,
    which then recorded their tracks on paper. Gait was recorded in terms
    of the tangent, that is the ratio of the width and length of step. At
    a body weight dosage of about 5 (mg/kg)/day the ratio was less than
    normal, a change the author attributed to an exaggeration of the
    stretch reflex. At dosages of about 10, 20, and 30 (mg/kg)/day, the
    ratio was progressively greater than the normal as a result of
    broadening of the gait and shortening of the steps. These same dosage
    levels did not affect problem-solving behaviour or speed of
    locomotion. The experimental animals were generally less reactive to
    stress than normal ones. Thus, the author attributed hyperirritability
    of rats poisoned by DDT to exaggerated motor responses.

        The major toxic action of DDT is clearly on the nervous system,
    and it requires an intact organism for full expression. The fact that
    DDT causes a myotonic response in muscle and substitution of a train
    of spikes for the normal diphasic electroneurogram (Eyzaguirre &
    Lilienthal, 1949) is in marked contrast with the absence of detectable
    injury or, in fact, any response in other isolated tissues. As early
    as 1945, Lewis & Richards (1945) found DDT to be inert when it was
    applied to tissue cultures of heart, kidney, stomach, intestine,
    liver, and muscle from 7 to 9-day chick embryos, and of brain and
    spleen from a one-day rat. The physiology of the cells including the
    mytoses of fibroblasts was normal. The migration and extension of the
    various cells was unchanged. The authors stated that "living
    fibrilloblasts, as they moved about in the cultures, sometimes touched
    or even migrated over DDT crystals without appreciable injury to
    themselves during a period of several days". Some observations were
    carried out for periods as long as 21 days.

        In spite of the importance of the nervous system, a detailed
    review of early literature indicates that, although the presence of
    some specialized nervous function may be necessary for the
    manifestation of DDT poisoning, the mere occurrence of specialized
    nerve fibres in certain protozoa or the occurrence of a rather complex
    nervous system in molluscs is not sufficient to render these forms
    susceptible. Just as there is no explanation for the effect of DDT on
    susceptible species, so there is no explanation for the fact that
    certain species and even entire phyla are inherently resistant to the
    compound.

        A review (Hayes, 1959) of literature on the effects of DDT on the
    nervous system reveals that all major parts, both central and
    peripheral, are affected. Whereas effects on specific portions,
    notably the cerebellum and the motor cortex, have been viewed as of
    greatest importance, it probably is more accurate to emphasize the
    interaction of functions, all modified to some degree.

        There is reason to think that the mechanism of action of DDT is
    its action on membranes in the nervous system, especially axonal
    membranes. Certainly action on membranes is a fundamental property of
    the compound. In fact the potassium conductance induced by valinomycin
    at 10-6 mol/litre in a synthetic lecithin-decane membrane is
    reversed by DDT at 3 × 10-6 mol/litre (Hilton & O'Brien, 1970).

        Attention was focused quite early on the effects of DDT on axonal
    membranes. Using the giant axons of the cockroach, Narahashi &
    Yamasaki (1960) showed that DDT prolonged the recovery phase of the
    action potential. They concluded that it slows the efflux of potassium
    ions from the axon. Later, using the voltage clamp technique and giant
    axons of the lobster, Narahashi & Haas (1967) showed that DDT, at a
    concentration of 5 × 10-4 mol/litre of bathing medium, prolonged the
    flow of sodium ions as well as interfering with the flow of potassioum
    ions: in other words DDT delayed shutting of the Na+ gate and
    prevented full opening of the K+ gate.

        Na+-, K+-, and Mg2+-adenosine triphosphatase (EC 3.6.1.3) is
    involved in ion transport in the nervous system. Matsumura & Patil
    (1969) showed that a preparation of this enzyme from a nerve ending
    fraction of the rabbit brain was inhibited by DDT at concentrations as
    low as 10-8 mol/litre. There was a good correlation between the
    degree of its inhibition by analogues of DDT and their toxicity to
    mosquito larvae. A similar enzyme that binds 14C-DDT has been
    isolated from the synapses of rat brain (Bratowski & Matsumura, 1972).
    Both the electrophysiological changes and the enzyme inhibition
    exhibit a negative temperature coefficient, an important feature of
    DDT poisoning in insects but not in mammals (Hoffman & Lendle, 1948;
    Deichmann et al., 1950).

        At a supralethal dosage of 600 mg/kg, DDT caused a marked decrease
    in the concentration of cortical and striatal acetylcholine and of
    brainstem norepinephrine in rats and a significant increase in
    brainstem 5-hydroxyindoleacetic acid. All of the neurotoxic signs of
    poisoning were blocked by  p-chlorophenylalanine, while other
    inhibitors blocked one or other, but not all of the effects. It was
    concluded that changes in the metabolism of 5-hydroxytryptamine and
    norepinephrine might be responsible for DDT-induced hyperthermia while
    acetylcholine might be related to tremors and convulsions (Hrdina et
    al., 1973). These and related matters have been reviewed in great
    detail by Hrdina et al. (1975). Apparently studies have not been made
    at a range of dosages that would make it possible to know whether
    these changes are a result or a cause of poisoning; the possible
    therapeutic effect of  p-chlorophenylalanine has also not been
    explored.

        It was reported by Haikina & Silina (1971) that administration of
    DDT to rats at only one-fifth of the LD50 for the 2 days increased
    the amount of 5-oxyindoleacetic acid excreted in urine by 188%. This
    indicates a change in the metabolism of serotonin, but its
    significance is not clear.

        One sensitive measure of brain activity is the electroencephalo-
    gram (EEG). Farkas et al. (1969) found that the EEG wave frequency
    increased considerably in resting rats that had received
    20 (mg/kg)/day of DDT as a result of dietary intake. Rats that had
    received either 5 or 10 (mg/kg)/day did not exhibit this change while
    at rest, but even those receiving 5 (mg/kg)/day exhibited this change
    when exposed to a rhythmic light stimulus. The EEG may become abnormal
    only a minute or two after administration of a large dose of DDT;
    4 stages of the electrical activity culminating in generalized
    seizures have been described by Joy (1973). Phenobarbital, but not
    phenytoin or trimethadione, was effective in stopping the seizures.

    7.1.2.1  Cause of death

        Death from DDT poisoning is usually the result of respiratory
    arrest. The heart continues to beat to the end and in some instances
    continues a little while after respiration stops. Deichmann et al.
    (1950) found that the onset of hyperirritability in rats was
    accompanied by an increase in the frequency and amplitude of
    respiration. Later, with the occurrence of tremors, the depth of
    respiration frequently returned to a more normal level, but the rate
    remained high. In some animals, respiration stopped suddenly after a
    deep inspiration during a tonic convulsion. In other animals, the rate
    and amplitude decreased progressively and finally ceased without any
    terminal spasm. Animals that die of respiratory failure caused by DDT
    do so after a relatively long period of muscular activity that leaves
    them exhausted.

        It was shown by Philips & Gilman (1946) and Philips et al. (1946)
    that the hearts of dogs given large intravenous doses of DDT were
    sensitized to epinephrine. This was true not only of injected
    epinephrine but also of the compound released by the adrenal glands
    during a seizure. Stimulated in this way, the sensitized hearts of
    dogs developed an irreversible, fatal ventricular fibrillation.
    However, the hearts of monkeys were able to recover from fibrillation
    and resume normal rhythm. It is not clear how important sensitization
    of the myocardium is when DDT is administered by other routes, but
    ventricular fibrillation may be the cause of death in animals that die
    suddenly, soon after onset of poisoning.

    7.1.2.2  Treatment of poisoning in animals

        Studies on the treatment of poisoning will be discussed in this
    section since all the more successful studies of treatment of animals
    poisoned by DDT involve the nervous system. Smith and Stohlman (1944)
    noted the possibility that narcotics in general, may exhibit an
    antagonism to DDT. Rats survived on a diet containing DDT at a
    concentration of 1000 mg/kg for 90 days when they received
    cyclohexanone in the same diet at the rate of 2000 mg/kg, but were
    uniformly killed in a shorter period when they received DDT at the
    same rate without cyclohexanone. Later, it was shown that
    cyclohexanone offers no protection when used as a solvent for single
    massive doses of DDT (Deichmann et al., 1950).

        Smith & Stohlman (1945) showed that, when given as required after
    the onset of illness, urethane and, to a lesser extent, phenytoin
    sodium protected rats from poisoning. Sodium amobarbital gave slight
    benefit, sodium phenobarbital a doubtful benefit, and paraldehyde no
    protection at all. All drugs were given intraperitoneally except
    paraldehyde, which was given by stomach tube. The mortality of rats
    treated with urethane was 12.5% and that of their controls was 80%. A
    total dosage of 1.2-2.5 mg/kg spread over a period of 1-3 days, was
    found most satisfactory. Phenytoin sodium gave a mortality of 46.7%,
    compared with 96.7% for the controls. The smallest effective dosage
    was 200-250 mg/kg, a value very close to the LD50 which, under the
    conditions of the test was 300 mg/kg.

        Lauger et al. (1945a, b) also found that sodium phenobarbital was
    of questionable value in treating rats poisoned by DDT. However,
    completely different results were seen in larger animals.
    Phenobarbital was, by far, the most outstanding remedy tested by
    Philips & Gilman (1946). In a dosage well below the anaesthetic level,
    it not only prevented death in many instances but also controlled
    tremor and convulsions. Signs of illness were more readily controlled
    in dogs and cats than in monkeys, which required nearly a full
    anaesthetic dosage before tremors completely disappeared.

        Magnesium sulfate did not reduce mortality in poisoned dogs and
    cats although it did control tremors and convulsions briefly. Sodium
    bromide was entirely ineffective. Mortality was reduced with urethane,
    but a full anaesthetic dosage was required to control tremor and
    convulsion. Similarly, sodium barbital and sodium pentobarbital
    controlled symptoms only when given in full anaesthetic doses and,
    even then, did not greatly reduce mortality. Phenytoin, when given to
    rats before they received DDT, reduced the lethal action without
    showing a notable effect on the signs of poisoning; phenytoin was not
    effective in cats.

        Vaz et al. (1945) were, apparently, the first to note the
    antidotal effect of calcium in DDT poisoning. Dogs were given DDT
    orally as a 10% oily solution at a daily dosage of 100 mg/kg until
    signs of intoxication appeared. The same dosage could then be repeated
    to produce intense symptomatology from which the animals would recover
    spontaneously in 12-24 h. For the actual tests, a larger challenge
    dosage of DDT (150 to 200 mg/kg) was used. Each dose of calcium
    gluconate (30 ml of a 10% solution) was injected intravenously into
    dogs weighing 8-18 kg. Dogs that were injected with calcium gluconate
    dally for 4 days and, challenged with a large dose of DDT on the
    fourth day, did not develop any symptoms or only slight ones. Dogs
    receiving a single dose of calcium gluconate showed symptoms of short
    duration and survived following a dosage of DDT large enough to kill 2
    controls.

        Cats poisoned by the intravenous injection of a soya-lecithin-corn
    oil emulsion of DDT were studied by Koster (1947). A comparison was
    made of several aspects of intoxication including number of
    convulsions, general severity (tremors, prostration, dyspnoea),
    duration, and mortality. Both calcium gluconate and sodium gluconate
    reduced mortality but not severity. Gluconic acid increased the
    survival time, reduced mortality, but did not reduce the number or
    severity of the convulsions. Calcium chloride reduced convulsions, but
    not mortality or tremors. Molecular equivalent doses of the candidate
    antidotes were used. Gluconic acid and its two salts were effective
    against an LD95 dosage of DDT. The life-saving capacity of calcium
    gluconate at a rate of 40 mg/kg was confirmed by Judah (1949) even
    though he found normal blood calcium values in most poisoned but
    unmedicated animals. One animal showed a high calcium value, and
    Cameron & Burgess (1945) reported a similar result. It has been
    suggested that increased blood calcium may be associated with acidosis
    caused by the accumulation of lactate.

        Thus, calcium has an antidotal action against DDT in intact
    animals of several species. The suppression by calcium of the effect
    of DDT on the isolated nerve and muscle of the rat has been
    demonstrated (Eyzaguirre & Lilienthal, 1949). The hypothesis has been
    advanced (Welsh & Gordon, 1946; Gordon & Welsh, 1948) that certain
    neurotoxins, including DDT, act by delaying the restoration of calcium

    ions to a surface complex, following breaking of the chelate linkage
    of calcium ions to surface polar groups by an initial exciting
    impulse. This action of the neurotoxin is conceived as depending
    largely on its physical rather than on its chemical properties. The
    hypothesis is helpful in explaining the fact that a wide variety of
    chemically unrelated compounds produce repetitive responses in
    excitable tissue and also the fact that many compounds that show a
    high toxicity for arthropods and mammals are fat-soluble and
    relatively inert chemically. It has been pointed out that this
    hypothesis postulates a very localized action of calcium at the nerve-
    cell membrane; the hypothesis is not inconsistent with the finding
    that the blood calcium of poisoned animals may be unchanged or even
    increased.

        Having observed the effect of DDT on the metabolism of glucose and
    glycogen, Lauger et al. (1945a, b) investigated the use of glucose as
    an antidote. All of 10 dogs given 2000 mg of DDT per kilogram body
    weight orally in the form of an oil solution died within 8-24 h. Five
    of 10 dogs treated with one or more, 20 ml doses of 20% glucose
    survived the same dosage of DDT. The glucose was given intravenously
    in most instances.

        Koster (1947) found that glucose given before or after an LD33
    dosage reduced convulsions and mortality and, when given before the
    poison, reduced tremors, prostration, and dyspnoea in cats. Glucose,
    unlike gluconic acid and its sodium and calcium salts, was ineffective
    against an LD95 dosage except to increase the time of survival.
    Insulin, given intra-muscularly 16-25 min before DDT, increased the
    survival time and the severity of poisoning but did not affect
    mortality or convulsions. When given 53-130 min before DDT, insulin
    reduced convulsions in animals which died but increased convulsions,
    tremors, and other disorders in the survivors.

        The failure of amino and sulfhydryl compounds to influence the
    action of DDT was noted by Von Oettingen & Sharpless, 1946). Likewise,
    the addition of 0.2% choline chloride to the diet of rats receiving
    repeated doses of DDT had no effect on the accumulation of lipids in
    their liver (Sarett & Jandorf, 1947).

        In summary, it would appear that sedatives, ionic calcium, and
    glucose or other ready sources of energy are useful in treating
    poisoning by DDT. Dogs and cats can be treated somewhat more
    successfully than rats, perhaps because the metabolism of larger
    animals is slower.

        Although respiration may be temporarily restored in animals
    poisoned by DDT, studies have not been made to determine whether
    respiratory arrest in this condition is truly reversible as it is in
    poisoning by certain organic phosphorus insecticides.

    7.1.3  Renal system

        No dysfunction of the renal system attributable to DDT has been
    found even in animals receiving dosages sufficient to cause
    dysfunction of the nervous system or striking morphological changes of
    the liver. It is true that mild to moderate morphological changes have
    been reported in the kidneys of animals that have received massive
    single doses or repeated doses; for example, fatty degeneration,
    necrosis, and calcification (Lillie et al., 1947; Stohlman & Lillie,
    1948) or slight brown pigmentation of the convoluted tubular
    epithelium (Fitzhugh & Nelson, 1947). However, it sometimes has
    happened that a complete absence of change in the kidney has been
    reported in connexion with other studies carried out in the same
    laboratories (Lillie & Smith, 1944; Nelson et al., 1944).

    7.1.4  Gastrointestinal tract, liver, and enzymes

        Large doses of DDT produce vomiting in species that can vomit.
    Only doses that produce rather severe poisoning lead to anorexia.

    7.1.4.1  Liver

        Large doses of DDT cause focal necrosis of liver cells in several
    species (Lillie & Smith, 1944; Nelson et al., 1944; Cameron & Burgess,
    1945; Lillie et al., 1947; Deichmann et al., 1950; Ortega et al.,
    1956). At least some investigators (Cameron & Burgess, 1945) have
    considered that the liver lesions produced by large dosages are
    sufficient to account for death. All have agreed that necrotic lesions
    occur only in connexion with potentially fatal dosages.

        A very different kind of liver change is produced in some rodents
    but not in other animals by small or moderate dosages of DDT. The
    biochemical aspects of these changes are discussed in section 7.1.4.2,
    while the morphological aspects are discussed in section 7.1.9.

    7.1.4.2  Microsomal enzymes of the liver

        All enzymes can be inhibited  in vitro and many of them can be
    inhibited  in vivo. The toxic action of a number of compounds is
    clearly the result of their inhibition of one or more enzymes. So far,
    only a few enzymes or enzyme systems are known to be induced by
    chemicals; the outstanding example is the induction of microsomal,
    mixed-function enzymes of the liver and some other organs that are
    produced in greater quantity in response to certain hormones and other
    normal constituents of the body (Conney, 1967), some foods
    (Wattenberg, 1971), or a wide range of drugs or other foreign
    chemicals. Such induction requires intact cells and does not occur
    when the inducer is brought in contact with the purified enzymes  in
     vitro. Microsomal enzymes were first recognized and studied in the
    liver, but they are now known to exist, generally in lower
    concentrations, in other tissues.

        Microsomal enzymes are known to be associated with oxidation ( N-,
     O-, and  S-dealkylation, deamination, epoxidation, disulfuration,
    hydroxylation) of both rings and side chains, oxidation of both
    nitrogen and sulfur, reduction of nitro groups and of azo compounds,
    hydrolysis, and conjugation. Most of the changes produced by
    microsomal enzymes render oil-soluble compounds more water-soluble
    and, therefore, more easily excreted. Mainly for this reason, most
    biotransformations promoted by microsomal enzymes are true
    detoxications. However, some of the reactions promoted by this system
    of enzymes render specific compounds more toxic.

        In the rat, DDT has been shown to promote the biotransformation of
    hexobarbital (Hart & Fouts, 1963), phenazone (Hart & Fouts, 1963;
    Kinoshita et al., 1966),  p-nitrobenzoic acid (Hart & Fouts, 1963),
    aniline (Hart & Fouts, 1963), dieldrin (Street, 1964; Street et al.,
    1966; Street & Chadwick, 1967; Pearl & Kupfer, 1971),  o-ethyl  o-4-
    nitrophenyl phenylphosphonothioate (EPN) (Kinoshita et al., 1966),
     p-nitroanisole (Kinoshita et al., 1966; Vainio, 1974), methyprylon
    (Datta & Nelson, 1968), meprobamate (Datta & Nelson, 1968),
    chlordizepoxide (Datta & Nelson, 1968), aldrin (Gillett, 1968),
    lindane (Chadwick et al., 1971b), phenylbutazone (Welch & Harrison,
    1966), pentobarbital (Fredricks et al., 1974), 3,4-benzpyrene (Vainio,
    1974), and  p-nitrophenol. The biotransformation of  p-nitrophenol
    involves conjugation by the microsomal enzyme uridinediphospho-
    glucuronyltransferase, and its demonstration requires activation of
    the microsomes, for example by trypsin (Vainio, 1974, 1975; Rantanen et
    al., 1975).

        In the mouse, DDT has been shown to promote the biotransformation
    of pentobarbital (Gabliks & Maltby-Askari, 1970).

        In the guineapig, DDT promoted the metabolism of dieldrin
    (Wagstaff & Street, 1970).

        DDT in the squirrel monkey promoted the metabolism of EPN and
     p-nitroanisole; the first required a DDT dosage of 5.0 (mg/kg)/day, but
    the latter required only 0.5 (mg/kg)/day (Cranmer et al., 1972). DDT
    did not promote the metabolism of 14C-DDT in squirrel monkeys
    (Chadwick et al., 1971a) but did promote the metabolism of
    phenylbutazone in the dog (Welch & Harrison, 1966) and the metabolism
    of estradiol in the pigeon (Peakall, 1970).

        In the chicken, DDT failed to affect  N-demethylase or the
    concentration of cytochrome P-450, and reduced aniline hydroxylase
    activity. The influences of dosage, duration of dosing species, and
    reproductive state on microsomal enzymes in birds are poorly
    understood (Sell et al., 1971).

         o,p'-TDE has been shown to promote the metabolism of
    pentobarbital in the mouse (Gabliks & Maltby-Askari, 1970),
    phenobarbital in the rat (Straw et al., 1965), and cortisol in the
    guineapig (Kupfer et al., 1964).

        The metabolism of DDT is promoted by DDT itself in the hamster but
    not in the mouse (Gingell & Wallcave, 1974).

        In rats, the metabolism of DDT is promoted by phenytoin (Cranmer,
    1970). The metabolism of DDT and DDE in cows is promoted by
    phenobarbital (Alary et al., 1971; Fries et al., 1971).

        DDE, whether fed directly or produced metabolically from DDT,
    appears to be more important than DDT in inducing microsomal enzymes.
    The tissue level of DDE necessary for enzyme induction is lower in the
    rat than in the quail (and presumably other birds). Thus Bunyan et al.
    (1972), using residues in the heart as an index, found a maximum
    increase in cyto-chrome P-450 per gram of liver and a maximum activity
    of aniline hydroxylase levels at DDE levels of approximately 3 mg/kg
    in rats and 40 mg/kg in quail. However, at any given dietary level,
    higher tissue levels were reached by quail than by rats so that the
    dosage responses of the two were similar.

        Different inducers may activate different enzymes and, therefore,
    different metabolic pathways. Pretreatment of rats with lindane caused
    them to metabolize a single dose of radioactive lindane 2.5 times more
    efficiently than controls, whereas pretreatment with DDT caused a
    3.5-fold increase in the metabolism of lindane. Furthermore, the DDT
    pretreatment was followed by a different proportion of radioactive
    metabolites with a predominance of tetrachlorophenols, especially
    2,3,4,5-tetrachlorophenol (Chadwick et al., 1971b).

        The enzymes of weanling rats are more subject to induction than
    those of adult rats, but there is no evidence of a lag-period in
    induction in adults (Chadwick et al., 1975).

    7.1.4.3  Enzymes of intermediary metabolism

        As discussed in section 7.1.2, there is some reason to think that
    DDT acts by influencing an enzyme critical to the function of
    neurones. It is certainly clear that many of the side-effects of DDT
    are the result of its induction of microsomal enzymes (see sections
    7.1.4.2 and 7.1.9). In addition, DDT has been shown  in vitro and
    sometimes  in vivo to influence some enzymes of intermediary
    metabolism and other miscellaneous enzymes. So far, evidence is
    lacking that the degree of this inhibition in the intact organism is
    sufficient to have any influence on function.

        The hyperglycaemia observed during much of the early part of acute
    poisoning may be associated with an increase in 4 gluconeogenic
    enzymes (pyruvate carboxylase (EC 6.4.1.1), phosphoenolpyruvate

    carboxykinase (EC 4.1.1.32), fructose 1,6-diphosphatase, and glucose
    6-phosphatase (EC 3.1.3.9)). Increases in these enzymes in the renal
    cortex of rats, observed after a single dose at a rate as low as
    100 mg/kg, were greater at a dose of 600 mg/kg. The reaction occurred
    in both adrenalectomized and normal rats (Kacew & Singhal, 1972).
    Similar responses were observed in the same enzymes in the liver
    following a single dose at the rate of 100 mg/kg or more or following
    45 daily doses at rates of 5 or 25 (mg/kg)/day. The changes are not
    mediated through a release of corticosteroids from the adrenal glands
    (Kacew & Singhal, 1973). The same authors (Hrdina et al., 1975;
    Singhal & Kacew, 1976) have reviewed the extensive evidence
    contributed mainly by themselves indicating that the changes in
    glucose homeostasis are mediated by stimulation of the cyclic
    adenosine monophosphate (AMP)-adenylate cyclase system in the liver
    and in the kidney cortex by  p,p'-DDT and a number of other organic
    chlorine pesticides. However, the smallest single dosage of  p,p'-DDT
    that produced a statistically significant change in the enzymes or in
    the production of cyclic AMP was 180 mg/kg and  o,p'-DDT was as
    effective as the  p,p'-isomer; these findings indicate that the
    carbohydrate changes are results not causes of poisoning.

        DDT and some other compounds induce increased activity of
    D-glucuronolacetone dehydrogenase (EC 1.1.1.70) in the supernatant
    fraction of rat liver, and this is consistent with evidence of
    increased D-glucaric acid in urine after dog treatment (Marselos &
    Hanninen, 1974). Urinary excretion of L-ascorbic acid is also
    increased by DDT, but apparently this is not caused by an increase in
    an enzyme producing this acid but rather by an increase in several
    microsomal and cytosol enzymes that contribute to an increase in free
    glucuronic acid from which L-ascorbic acid is formed (Rantanen et al.,
    1975).

        A review (Hayes, 1959) of early literature indicates that high
    concentrations of DDT inhibit phosphatidase (EC 3.1.1.4), muscle
    phosphatases, carbonic anhydrase (EC 4.2.1.1), oxalacetic carboxylase
    (EC 4.1.1.3), and increase the activity of cytochrome oxidase
    (EC 1.9.3.1) and succinc dehydrogenase (EC 1.3.99.1). However, none of
    these changes with the possible exception of inhibition of carbonic
    anhydrase could be shown to have any connexion with the toxic action
    of DDT or even with its side-effects. Neal et al. (1944) reported a
    small but consistent increase in the volume of urine excreted in 24 h
    when dogs were dosed orally or by insufflation at the rate of 100
    (mg/kg)/day. No other change in the urine and no change in kidney
    function was demonstrated. The possibility that increased urinary
    output is related to the inhibition of carbonic anhydrase (Torda &
    Wolff, 1949) may deserve attention. However, re-examination of data
    from volunteers receiving 3.5 or 35 mg/man per day did not indicate
    any increase in urinary volume compared with controls (Hayes et al.,
    1971).

        It has been claimed (Keller, 1952) that DDT inhibits carbonic
    anhydrase in bovine erthrocytes. However, the method was criticized by
    Dvorchik et al. (1971), who found  in vitro inhibition only by
    concentrations of DDT, unlikely to be survived by living animals. Far
    more attention has been given to inhibition of carbonic anhydrase in
    the shell gland of birds than in erythrocytes, and it has been
    suggested (Bitman et al., 1970; Peakall, 1970) that inhibition of the
    shell gland enzyme is an explanation for eggshell thinning in certain
    birds. However, the same criticism holds for the shell gland; there is
    no evidence that the degree of inhibition reported interferes with
    function.

        On the other hand, many enzymes including plasma amylase, aldolase
    (EC 4.1.2.13), glutamic-pyruvic transaminase (EC 2.6.1.2), and
    isocitric dehydrogenase (EC 1.1.1.41) were not changed in squirrel
    monkeys given dosages ranging from 0.05 to 50 (mg/kg)/day; the highest
    dosage proved fatal within 14 weeks (Cranmer et al., 1972).

    7.1.5  Cardiovascular system

        Most dogs, killed by a single dose of DDT, die of ventricular
    fibrillation, and the same is true of some cats, monkeys, and rabbits
    (Philips & Gilman, 1946). At any given dosage of DDT, ventricular
    fibrillation is more likely to occur if the animal receives exogenous
    epinephrine or is stimulated in such a way that the compound is
    released by the adrenals. Besides fibrillation, dogs may exhibit
    extrasystoles and changes in the T-wave (Philips et al., 1946).
    Monkeys differ from dogs in that the DDT-sensitized heart is able to
    recover from fibrillation and resume a normal rhythm (Philips et al.,
    1946).

        Thus, DDT not only sensitizes the myocardium in a way similar to
    that of halogenated hydrocarbon solvents, but, through its action on
    the central nervous system, produces the stimulus that increases the
    likelihood of fibrillation.

        There is no evidence that repeated, tolerated doses of DDT
    sensitize the heart. Rats were fed DDT at a dietary level of about
    10 (mg/kg)/day for 8 months, during which time they received weekly,
    intraperitoneal doses of vasopressin, a compound that causes a
    temporary myocardial ischaemia. Electrocardiograms did not show any
    significant increase in cardiac arrhythmias in the DDT-fed rats
    compared with controls. Intravenous noradrenalin given at the end of
    the 8-month period did not produce a greater incidence of arrhythmias
    in the DDT-fed rats. The same results were obtained in rabbits treated
    in essentially the same way (Jeyaratnam & Forshaw, 1974).

         TDE. The main action of TDE, and especially of  o,p'-TDE, is on
    the liver with secondary effects on the adrenals in dogs and perhaps
    in other species. However, Cueto (1970) showed that at a dosage of
    50 (mg/kg)/day for 14 days,  o,p'-TDE caused a gradually progressive

    hypotensive failure in dogs injected with epinephrine or
    norepinephrine, while leaving the cardioaccelerator and immediate
    pressor response to these drugs unchanged. The hypotensive failure was
    associated with weakening of the contractile force of the heart and
    with a reduction of plasma volume. The latter may have been caused by
    a loss of fluid from the intravascular compartment and was not caused
    by a release of histamine. The hypotensive state could be prevented to
    a significant degree by pretreatment with prednisolone.

    7.1.6  Respiratory system

        The effects of DDT on the respiratory system are secondary to
    effects on the nervous system and are discussed in section 7.1.2.1.

    7.1.7  Reproductive system

        It was shown very early (Burlington & Linderman, 1950) that DDT
    produces a striking inhibition of testicular growth and secondary
    sexual characteristics of cockerels, when injected subcutaneously in
    dosages as high as 300 (mg/kg)/day. Changes in the testis involve the
    tubules, and not the interstitial tissues, and they have been
    attributed to an estrogen-like action of DDT.

        It must be noted that the action of DDT on the testis of the
    chicken is dosage-related. Before the problem of residues became
    evident, DDT was used extensively for control of lice and common mites
    on chickens without any adverse effects on egg production or other
    aspects of reproduction. Many rats would be killed the first day if
    they were given the dosage of DDT that has been shown to affect the
    testis in cockerels. The report that, under special conditions, DDT
    has it gonadotoxic effect (Rybakova, 1968) is of questionable
    significance in view of the results of multigeneration tests in rats,
    mice, and dogs.

        Ottoboni (1969) found that female rats reproduced normally when
    fed DDT for two generations at dietary levels as high as 200 mg/kg
    (about 10 (mg/kg)/day, except during lactation when intake was
    increased about 3-fold). In fact, at a dietary level of 20 mg/kg, the
    dams had a significantly longer reproductive life span (14.55 months)
    than did their littermate controls (8.91 months); the number of
    females becoming pregnant after the age of 17 months and the number of
    successful pregnancies after that age differed significantly between
    the two groups (Ottoboni, 1972).

        In a study focused mainly on DDT in milk, the ability of rats to
    reproduce at a dietary level of 200 mg/kg was confirmed, and the
    ability of dams, injected intraperitoneally at levels as high as
    100 (mg/kg)/day, to rear their young was demonstrated (Hayes, 1976b).

        A six-generation test of reproduction in mice did not show any
    effect of DDT at a dietary level of 25 mg/kg on fertility, gestation,
    viability, lactation, and survival. A dietary level of 100 mg/kg
    produced a slight reduction in lactation and survival in some
    generations but not all, and the effect was not progressive. A level
    of 250 mg/kg was distinctly injurious to reproduction (Keplinger et
    al., 1970). The dietary concentrations used equalled dosages of 3.33,
    13.3 and 33.2 (mg/kg)/day in nonpregnant, non-lactating, adult female
    mice. The intake is much higher in both young and lactating mice. The
    authors concluded that their study provided no obvious reason for
    continuing reproduction tests for more than three generations.

        Four female dogs of unstated age that had previously received DDT
    at the rate of 12 (mg/kg)/day, 5 days a week, for 14 months were mated
    when they went into heat. The males involved had been fed aldrin
    (0.15 (mg/kg)/day) plus DDT (60(mg/kg)/day) for 14 months prior to
    breeding but not during breeding. Two of the females went into heat
    but failed to become pregnant, and one failed to come into heat during
    12 months after feeding stopped. Four of 6 pups born to the fourth
    female died within one week of birth; the other 2 were weaned
    successfully even though only 2 posterior mammae of the mother were
    functional (Deichmann et al., 1971b). A 3-generation study failed to
    confirm any of the injuries suggested by the study of 4 dogs. In the
    3-generation study, male and female dogs were fed technical DDT from
    weaning at rates of 0, 1, 5, and 10 (mg/kg)/day. Observations were
    made on 135 adult females, 63 adult males and 650 pups. There were no
    statistically significant differences between controls and DDT-treated
    dogs in length of gestation, fertility, success of pregnancy, litter
    size, lactation ability of the dams, viability at birth, survival to
    weaning, sex distribution, growth of pups, morbidity, mortality,
    organ/body weight ratios, or gross histological abnormalities in all
    the animals studied. The only clear difference was that DDT-treated
    females had their first estrous 2 or 3 months earlier than the control
    animals. There was a slight increase in liver/body weight ratio in
    some DDT-treated animals but the difference was not statistically
    significant, dosage related, or associated with any histological
    change (Ottoboni et al., 1977).

         o,p'-DDT. Intraperitoneal injection of technical DDT at a dosage
    as low as 5 mg/kg or of  o,p'-DDT at 1 mg/kg caused a significant
    increase in the weight of the uterus of normal, immature female rats
    or of ovariectomized adult females. Stimulation caused by  p,p'-DDT
    was much less. Treatment of rats with DDT, especially  o,p'-DDT, 2 h
    before injection of estradiol-17-6,7-3H inhibited uptake of the
    hormone by the uterus  in vivo, possibly by competition for binding
    sites. Isomers of TDE and DDE do not influence uterine weight or the
    binding of estradiol (Welch et al., 1969). It seems unlikely that
    metabolic activation of  o,p'-DDT is necessary, as is true of  o,p'-
    methoxychlor. The binding and estrogenic activity of DDT analogues in
    rats is only about 1/10 000 as great as that of diethylstilbesterol
    (Nelson, 1973).

        A considerably smaller dosage of  o,p'-DDT resulting from a
    dietary level of 10 mg/kg for 2-9 months did not have any effect on
    reproduction in ewes (Wrenn et al., 197 lb). In a similar way, dietary
    levels of  o,p'-DDT as high as 40 mg/kg, giving a dosage level of
    about 2.1 (mg/kg)/day in rats, failed to interfere with reproduction
    and lactation in these animals although dosage was continued
    throughout 2 pregnancies (Wrenn et al., 1971a).

        The report (Heinrichs et al., 1971) that  o,p'-DDT significantly
    advances puberty, induces persistent vaginal estrus after a period of
    normal estrus cycles, and causes other reproductive abnormalities in
    female rats would appear at first to be inconsistent with the lack of
    effect of technical DDT or of  o,p'-DDT on reproduction cited above.
    The same is true of other effects of  o,p'-DDT demonstrated by the
    same investigators (Gellert et al., 1972). The abnormal effects were
    obtained initially by injecting 1 mg of the  o,p'-DDT subcutaneously
    on the second, third, and fourth days of life (counting the day of
    birth as zero). Because rat pups on the third day weigh about 12 g or
    less each, it follows that the subcutaneous dosage was about
    83.3 (mg/kg)/day or more, that is about 40 times greater than the
    highest oral dosage of  o,p'-isomer fed to breeding rats and about
    105 times greater than the levels ingested by the general population
    with their food.

        Because of its estrogenic properties, DDT was considered as a
    possible cause of abortion in dairy cattle, but no evidence of a
    relationship was found (Macklin & Bibelin, 1971).

    7.1.8  Endocrine organs

        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 of 2 isomers of TDE are now
    considered to be mainly secondary to the induction of microsomal
    enzymes of the liver in most species.

         TDE. TDE (DDD) is an insecticide in its own right as well as a
    metabolite of DDT. The compound is used as a drug to control different
    forms of adrenal overproduction of corticoids in man (see section
    8.2.8). This therapy was originally based on the demonstration that
    DDD (Nelson & Woodard, 1948, 1949) and especially  o,p'-TDE (Cueto &
    Brown, 1958) caused gross atrophy of the adrenals and degeneration of
    the cells of its inner cortex in dogs. This is true even though it was
    originally reported (Nelson & Woodard, 1948, 1949) that TDE produced
    almost no detectable damage to the adrenals of rats, mice, rabbits,
    and monkeys, and the finding was confirmed and extended by other
    investigators to other species, including man (Zimmerman et al.,
    1956). In the dog,  o,p'-TDE produced gross atrophy of the adrenals,
    when administered at a dosage of only 4 (mg/kg)/day. The dosage of
    technical grade TDE required to produce the same effect was

    50-200 (mg/kg)/day (Cueto & Brown, 1958). However, in spite of its
    exceptional susceptibility, there is a definite threshold below which
    the dog does not respond. About 15% of technical DDT is  o,p'-isomer,
    much of which is gradually metabolized to  o,p'-TDE ( o,p'-DDD). Yet
    dogs remained healthy and reproduced normally in a 3-generation study
    involving dosages of technical DDT as high as 10 (mg/kg)/day (see
    section 7.1.7).

        It has recently been shown that, following massive dosage
    (60 mg/kg, administered intravenously), all of the isomers of TDE
    inhibited ACTH-induced steroid production in the dog, but the
    inhibition reached 50% of the control only 27 min after dosing with
    the  m,p'-isomer compared with 87 min with the  o,p'-isomer and
    4-18 h with the  p,p'-isomer. There was marked temporal correlation
    between the percentage inhibition of adenocorticotropic hormone
    (ACTH)-induced steroid production, the disruption of normal cellular
    structure of the fascicular and reticular zones of the adrenal cortex,
    and the severity of the damage to mitochondria in these zones caused
    by the 3 isomers (Hart et al., 1973). The effectiveness of  m,p'-TDE
    for treating metastatic adrenocortical carcinoma had already been
    demonstrated (Nichols et al., 1961), but it cannot be said that its
    value for this purpose has been compared adequately with that of
     o,p'-TDE. Furthermore, no effort has apparently been made to compare
    the effect of small daily doses of the 2 isomers in dogs.

        Like other organochlorine insecticides,  o,p'-TDE stimulates
    hepatic microsomal oxygenation of both drugs and steroids and,
    according to a very thorough review by Kupfer (1967), this may explain
    much of its action on corticoid metabolism in a wide range of species.
    Increased breakdown is indicated by increased excretion of polar
    metabolites, while nonpolar metabolites remain stable or even decrease
    -- a finding recently encountered in human patients (Hellman et al.,
    1973). However, the demonstrated effect on corticoid metabolism fails
    to explain why  o,p'- and  m,p'-TDE are unique in their overall
    effects on the adrenals, including their ability to produce
    adrenocortical atrophy in the dog. Other powerful inducers of
    microsomal enzymes lack these effects. It is clear that a reduction of
    steroid production accompanies atrophy of the adrenals of the dog. The
    review already cited (Kupfer, 1967) considers: (a) reduced steroid
    production in species other than the dog, including the possibility
    that such reduction is secondary to inhibition of glucose-6-phosphate-
    dehydrogenase (EC 1.1.1.49) activity in the adrenals and (b) blockage
    of steroid action by asteroid metabolite formed under the influence of
    DDD. However, the existence of these effects, much less their
    importance, remains obscure.

         o,p'-DDT. Oral administration of  o,p'-DDT to dogs at a rate of
    50 (mg/kg)/day stimulated the microsomal enzymes of the liver as
    indicated by increase in liver size, total protein, microsomal
    protein, and cytochrome P-450 concentration and by direct measurements
    of enzyme activity. These changes in the liver were accompanied,

    initially, by an increase in the size of the adrenals and of the cells
    of the zona fasciculata; these cells became vacuolated and devoid of
    acidophilic cytoplasm, and their nuclei became hyperchromatic and
    often peripheral in position. Synthesis of corticosteroids by the
    adrenal was not blocked (Copeland & Cranmer, 1974). Thus the effect of
    a substantial dosage of  o,p'-DDT was quite different from that of
     o,p'-TDE (DDE), although part of the metabolism of  o,p'-DDT must
    be by the same route.

    7.1.9  Carcinogenicity

        There is no doubt that DDT and a number of other organochlorine
    insecticides cause marked changes in the liver in various rodents and
    that these changes progress to tumour formation in some species,
    notably the mouse. There is serious disagreement as to whether the
    mouse tumours are malignant. Regardless of their nature, there is
    virtual certainty that they are peculiar to rodents and, therefore,
    interpretation of their significance for man or useful animals is
    difficult.

        Evidence for the carcinogenicity of DDT and its metabolites has
    been reviewed by the International Agency for Research on Cancer
    (IARC, 1974). Most of the experimental results are summarized in Table
    17. The conclusions of the IARC were that: (a) the hepatocarcino-
    genicity of DDT administered by the oral route has been demonstrated in
    several strains of mice, and shows a dosage-response relationship; (b)
    a dietary level of 2 mg/kg (above 0.3 (mg/kg)/day) produces a
    significant increase in hepatomas in male but not in female CFl mice
    and not in either sex of BALB/c mice; (c) increased incidence of
    tumours has been reported in some other organs of mice but not
    confirmed in multigeneration studies using a wide range of dosages;
    (d) evidence for the carcinogenicity of DDT in rats is not convincing
    and is negative in hamsters even at the higher dietary levels that
    they tolerate in comparison with rats and mice; (e) negative results
    in dogs and monkeys are inconclusive because of the small groups
    studied and the short duration of treatment; (f) liver cell tumour
    induction in trout is inconclusive because of a lack in control of the
    diet; and (g) the carcinogenicity of  p,p'-DDE is similar to that of
    DDT, but TDE produces a significant incidence of lung tumours.

        Actually, the number of dogs and monkeys was not small compared
    with similar studies on other chemicals. In an investigation on
    monkeys, dosing at lower levels was continued for 7.5 years. In both
    dogs and monkeys, dosages sufficient to cause liver damage, death, and
    neurological indications of DDT poisoning were included in the
    protocols (see Table 17). Apparently no new studies on dogs and
    monkeys have been reported. On the other hand, a new study on rats
    (Rossi et al., 1977) has given definite evidence of the tumorigenicity
    of DDT and phenobarbital, confirming the conclusion of Fitzhugh &
    Nelson (1947) which was based on less extensive data.

        In mice, liver tumours similar to those caused by DDT (see
    Table 17) have been reported in connexion with DDE and DDD (Tomatis &
    Turusov, 1975), chlorobenzilate, HCH, aldrin, dieldrin, mirex, and
    terpene polychlorinates (Tomatis et al., 1973). Similar tumours have
    been caused by the important drug, phenobarbital (Wright et al., 1972;
    Peraino et al., 1973; Thorpe & Walker, 1973; Ponomarkov et al., 1976).
    TDE also caused lung tumours (Tomatis et al., 1974a).

        The IARC review did not discuss the controversy over the nature of
    the tumours induced by DDT in the livers of some rodents, and it did
    not consider the relationship of these tumours to the induction of
    microsomal enzymes. The following paragraphs are concerned with these
    matters and specifically with those inducers that behave like DDT. The
    biochemical pattern of induction of mixed function oxidase enzymes is
    similar for DDT and phenobarbital but distinctly different for
    3-methylcholanthrene (Vainio, 1975). Thus 3-methylcholanthrene and
    compounds like it must be excluded from the discussion. Similarly, the
    high degree of correlation between the ability of compounds to induce
    parenchymal liver tumours in mice and their ability to induce tumours
    in the liver and other organs of rats and hamsters cannot be accepted
    uncritically. As demonstrated in a study by Tomatis et al. (1973),
    this correlation is extremely good for compounds that are, or are
    suspected of being, carcinogens in man. However, the correlation is
    poor for organochlorine insecticides. In fact, the only one of these
    compounds that has increased the incidence of a tumour in another
    species is DDT, which induced liver tumours in rats in one experiment.

        The early morphological response of the rodent liver to DDT is
    similar to its response to moderate dosages of HCH, chlordane,
    dieldrin, camphechlor (Toxaphene) (Lehman, 1952; Ortega et al., 1956),
    and the important drug, phenobarbital (Wright et al., 1972; Thorpe &
    Walker, 1973). The earliest change involves so much increase in the
    smooth endoplasmic reticulum of individual liver cells that they
    enlarge, and the large granules that are ordinarily scattered
    throughout the cytoplasm are displaced to the periphery of the
    affected cell. Quite early, some of the endoplasmic reticulum forms
    whorls that may have fat droplets as their centres -- thus justifying
    the term "lipospheres", applied to them by Ortega et al. (1956).
    Others have referred to these inclusions as "hyaline oxyphil masses"
    (Lillie & Smith, 1944) or "lamellar bodies" (Ito et al., 1973). These
    changes are accompanied by some increase in fat droplets, not all of
    which become surrounded by endoplasmic reticulum. This combination of
    changes (hypertrophy, margination, and lipospheres) is characteristic
    of rodents and of compounds that induce microsomal enzymes. Certain
    other changes have been reported but not confirmed. These include
    enlargement and morphological changes of the mitochondria (Obuchowska
    & Pawlowska-Tochman, 1973; Watari, 1973), increased numbers of primary
    lysosomes, and atrophy of the Golgi body (Watari, 1973), none of which
    were found by Ortega (1966).

        Table 17.  Effect on various animals of repeated oral doses of DDT

    Dosage                            Species  Duration    Results                                                       Reference
                                                                                                                     
    Range        Method and                                Animals                Mortality  Other
    (mg/kg)/day  concentration                             per test               (%)
                                                           (No.)

    41-80        800 mg/kg in diet    rata     2 years     36 males, 24 females              Increased mortality,        Fitzhugh & Nelson, 1947
                                                                                             typical liver changes,
                                                                                             liver tumours
                 46 mg/kg then        mouseb   18 months   36 males, 36 females              Hepatomas in 51 and         Innes et al., 1969
                 140 mg/kg in diet                                                           21% of males and females
                                                                                             respectively compared with
                                                                                             18 and 0.6% of controls
                 3200 mg/kg in diet   dog      39-49       10                     100        Liver damage, no            Lehman, 1965
                                               months                                        tumours
                 5000 mg/kg in diet   monkey   70 days     1 male                 100        Fatal poisoning             Durham et al., 1963

    21-40        400 mg/kg in diet    rata     2 years     24 males, 12 females              Increased mortality,        Fitzhugh & Nelson, 1947
                                                                                             typical liver changes
                 500 mg/kg in diet    rat      2.9 years   37 males, 35 females              Liver tumours in 45%        Rossi et al., 1977
                 250 mg/kg in diet    mousec   2           103 males, 90 females             Risk of liver tumour        Tomatis et al., 1972
                                               generations                                   increased 3.7 and 18.5
                                                                                             times in males and females,
                                                                                             respectively
                 250 mg/kg in diet    mousec   2           31 males, 121 females             Liver tumours in 48         Terracini et al., 1973
                                               generations                                   and 59% of males and
                                                                                             females, respectively
                 2000 mg/kg in diet   dog      39-49       4                      25         Minor liver damage          Lehman, 1965
                                               months                                        but no tumours

    Table 17 (Cont'd)

    Dosage                            Species  Duration    Results                                                       Reference
                                                                                                                     
    Range        Method and                                Animals                Mortality  Other
    (mg/kg)/day  concentration                             per test               (%)
                                                           (No.)

    11-20        100 mg/kg in diet    moused   2 years     100 males, 100 females            Hepatomas increased         Fitzhugh, 1970
                                                                                             in females of one strain
                                                                                             but no increase in
                                                                                             hepatocarcinomas

                 100 mg/kg in diet    mousec   2 years     30 males, 30 females              Risk of liver tumours       Walker et al., 1973
                                                                                             increased 4.4 times
                 100 mg/kg in diet    mousec   2 years     30 males, 3 females               Risk of liver tumours       Thorpe & Walker, 1973
                                                                                             increased 3.3 and 4.2 times
                                                                                             in males and females
                                                                                             respectively

    5-10         50 mg/kg in diet     mousec   2           127 males, 104 females            Risk of liver tumours       Tomatis et al., 1972
                                               generations                                   increased 2.45 and 3.46
                                                                                             times in males and females,
                                                                                             respectively
                 50 mg/kg in diet     mousec   2 years     30 males, 30 females              Risk of liver tumours       Walker et al., 1973
                                                                                             increased 2.9 times
                 400 mg/kg in diet    dog      39-49       2                      0          No effect                   Lehman, 1965
                                               months

    Table 17 (Cont'd)

    Dosage                            Species  Duration    Results                                                       Reference
                                                                                                                     
    Range        Method and                                Animals                Mortality  Other
    (mg/kg)/day  concentration                             per test               (%)
                                                           (No.)

    2.6-5        20 mg/kg in diet     mousee   2           48 males, 128 females             No increase in tumours      Terracini et al., 1973
                                               generations
                 200 mg/kg in diet    monkey   3-7.5       5 males, 5 females                No toxic effect             Durham et al., 1963
                                               years

    1.26-2.5     10 mg/kg in diet     mousec   2           104 males, 124 females            Risk of liver tumour        Tomatis et al., 1972
                                               generations                                   increased 2.26 and 2.46
                                                                                             times in males and females,
                                                                                             respectively

    0.626-1.25   25 mg/kg in diet     rat      2 years                                       No clinical effect;         Treon & Cleveland, 1955
                                                                                             males survived
                                                                                             longer than controls
                 50 mg/kg in diet     monkey   1.6 years   4 males, 1 female                 No toxic effect             Durham et al., 1963

    0.3126-      10 mg/kg in diet     rat      2 years                                       Typical liver changes;      Fitzhugh, 1948
    0.625                                                                                    no effect on
                                                                                             reproduction
                 12.5 mg/kg in diet   rat      2 years                                       No effect                   Treon & Cleveland, 1955
                 2.8-3.0 mg/kg in     mousee   5           683                               Tumours in 28.7% in         Tarjan & Kemeny, 1969
                 diet                          generations                                   controlsf

    0.15626-     2 mg/kg in diet      mousee   2           124 males, 111 females            Risk of liver tumour        Tomatis et al., 1972
    0.3125                                     generations                                   doubled in males,
                                                                                             unchanged in females
                 2 mg/kg in diet      mousee   2           58 males, 135 females             No increase in tumours      Terracini et al., 1973
                                               generations

    Table 17 (Cont'd)

    Dosage                            Species  Duration    Results                                                       Reference
                                                                                                                     
    Range        Method and                                Animals                Mortality  Other
    (mg/kg)/day  concentration                             per test               (%)
                                                           (No.)

    0.078126-    2.5 mg/kg in diet    rat      2 years                                       No effect                   Treon & Cleveland, 1955
    0.15625      5 mg/kg in diet      monkey   1.4-7.5     5 males                           No toxic effect             Durham et al., 1963
                                               years

    a  Osborne-Mendel.                                             e  BALB/c mice.
    b  Both (C57BL/6 × C3H/Anf) FI and (C57BL/6 × AKR) FI mice.    f  Lung carcinoma in 16.9% to 1.2% in controls; lymphomas 4.8% compared to
    c  CFI mice.                                                      1.0% in controls; leukaemias 12.4% and 2.6%; other tumours 5.8% and 1.0%,
    d  BALB/cJ and C3HeB/Fe J.                                        respectively.

        The characteristic changes develop promptly. An increase in smooth
    endoplasmic reticulum and the appearance of lamellar structures have
    been seen as early as 4 and 7 days, respectively, after the beginning
    of dosing (Wright et al., 1972).

        Although microsomal enzymes may be induced in other species,
    morphological changes in the liver, as viewed by the light microscope
    are not the same (Laug et al., 1950; Lehman, 1952; Ortega et al.,
    1956), or occur to lesser degree as viewed by the electron microscope
    (Wright et al., 1972).

        The changes in liver cells that characterize the induction of
    microsomal enzymes in rodents are distinct from the focal necrosis
    that may be produced with about the same ease in the livers of rodents
    or other species by fatal or near fatal dosages of organochlorine
    insecticides. The necrotic lesions do not progress because, if such
    high dosages are continued the animal dies, and, if dosing is stopped
    and the animal survives, the necrotic cells are removed by autolysis
    and phagocytic action and the lesions usually heal without scarring
    (Cameron & Burgess, 1945). Nevertheless, some scarring was found by
    Lillie & Smith (1944) and Lillie et al. (1947).

        At least in the early stages, the changes in liver cells that
    characterize induction of microsomal enzymes in rodents are reversible
    (Fitzhugh & Nelson, 1947; Ortega et al., 1956; Wright et al., 1972).
    The reversibility does not depend on cell removal but simply on
    reversion of the physiological and morphological condition of the
    cells to their original condition.

        Of course, reversibility is incompatible with progression, but
    whether observed irreversibility will be associated with progression
    must be determined directly in each instance. In the following
    paragraphs, the question of progression is discussed only after
    consideration of the problem of irreversibility in general.

        If dosing with organochlorine insecticides or other inducers is
    continued long enough and at a sufficiently high level, the liver
    changes become irreversible, if for no other reason than that the
    remaining life span of the animals is too short to permit excretion of
    the inducing chemical or complete reversion of the liver cells to
    their original state. The stage at which this shift to irreversibility
    occurs remains unknown, but it seems very likely that dosages
    sufficient to produce irreversible morphological change also exceed
    the physiological adaptability of the liver. The important distinction
    between adaptation and injury in relation to enzyme induction has been
    studied using dieldrin.

        Although some persons have tended to view even moderate
    enlargement of the liver or of individual liver cells as an injury,
    the evidence is strong that these changes are usually adaptive and

    beneficial to the organism, when they are the result of an increase of
    smooth endoplasmic reticulum and an associated increase in the
    activity of liver microsomal enzymes (Barka & Popper, 1967). However,
    it is obvious that any stimulus or effect may be harmful, if
    excessive. Hutterer et al. (1968) demonstrated that a distinction may
    be drawn between adaptation to dieldrin and decompensation resulting
    from excessive doses of it. Some of these authors, including Popper,
    showed that the same distinction could be drawn in connexion with
    other sources of potential liver injury.

        It was found that daily intraperitoneal administration of dieldrin
    to rats at the rate of 2 mg/kg produced enlargement of the liver,
    hypertrophy of the smooth endoplasmic reticulum, increases in
    microsomal protein and P-450 haemoprotein, and associated increases in
    the activity of microsomal enzymes; however, normal activity of other
    enzymes not derived from the microsomes was maintained. The activity
    of microsomal enzymes per mole of available P-450 haemoprotein
    remained unchanged. The highest level of activity of the processing
    enzymes was reached after 14 days, after which the new steady state
    was maintained. Rats that had received dieldrin at a rate of 2 mg/kg
    per day for 28 days were more tolerant to dieldrin than normal rats,
    as shown by the fact that they survived 25 consecutive daily doses at
    the rate of 5 mg/kg, a dosage that produced 70% mortality in
    previously untreated rats. In spite of the ability of the rats,
    pretreated with a moderate dosage of dieldrin, to survive a large
    dosage, their livers showed definite indications of decompensation in
    response to the high dosage. Although the smooth endoplasmic reticulum
    remained hypertrophic and the microsomal protein and P-450
    haemoprotein concentrations remained elevated, the enzyme activities
    per mole of available P-450 haemoprotein decreased, as did the
    activity of some enzymes not associated with microsomes. Much of the
    excess smooth endoplasmic reticulum formed tightly packed clusters of
    tubular membranes with no glycogen and little hyaloplasm, and some of
    the mitochondrial membranes were injured. It was suggested that the
    phase of decompensation represented by hypertrophic but hypoactive
    smooth endoplasmic reticulum might serve as a sensitive criterion of
    toxic injury before microscopic changes of a clearly harmful sort
    become recognizable (Hutterer et al., 1968).

        Other studies indicating not only the presence of adaptive change
    over a range of dosages but also the failure of adaptation and onset
    of injury at sufficiently high dosage levels have been reported for
    butylated hydroxy-toluene (Gilbert & Golberg, 1967) and for DDT
    (Hoffman et al., 1970). Hoffman and his colleagues found that, when
    DDT was fed to male weanling rats for only 14 days at dietary
    concentrations of 0.5 to 2048 mg/kg, concentrations of 0.5 and 2 mg/kg
    had no effect on the  O-demethylation reaction used as a test, but
    concentrations of 4-750 mg/kg produced increases in the rate of
    metabolism, proportional to the log of dosage. Extrapolation of this
    portion of the dosage response curve to the abscissa provided a
    calculated no-effect level of 3.27 mg/kg equivalent to about

    0.327 (mg/kg)/day. This is in reasonable agreement with other
    estimates of the threshold for induction of various enzymes in the
    rat, including some studies involving longer administration of DDT.
    These estimates, expressed as (mg/kg)/day, are approximately 0.05
    (Kinoshita et al., 1966; Street et al., 1969), 0.5 (Schwabe &
    Wendling, 1967) and 0.125 (Gillett, 1968). All of the estimates are of
    the same order of magnitude as the 0.25 (mg/kg)/day known to be
    effective in man (Laws et al., 1967; Poland et al., 1970), but all are
    over 100 times greater than the highest dosage received by members of
    the general population during the late 1960s (Duggan, 1968).
    Increasing the dietary level to more than 750 mg/kg did not produce
    any further increase in enzyme activity. Intake of less than 128 mg/kg
    did not produce any increase in liver weight, within the period of
    observation; increase was proportional to dosage within the range of
    128 to 512 mg/kg and was submaximal at intakes above 512 mg/kg.

        Some other compounds, notably phenobarbital, produced
    morphological changes in the liver similar to those produced by some
    organochlorine insecticides (Wright et al., 1972; Thorpe & Walker,
    1973). It seems possible that sufficiently high doses of
    phenobarbital, for example, may lead to a failure of adaptation and to
    levels of enzyme activity that do not correspond to dosage.

        As indicated above, the earliest morphological changes caused by
    enzyme inducers in the rodent liver involve separate cells in the
    centrolobular area. If the dosage is sufficiently high and prolonged,
    nodules consisting entirely of hypertrophied cells may appear. At
    first, these microscopic nodules are distinguishable only by pattern;
    they have no bounding membrane and they do not compress or change in
    any other detectable way the smaller liver cells that surround them.
    Some nodules may become large enough to be seen without a microscope,
    and a few may exceed 1 cm in diameter. In these large nodules, there
    is almost complete loss of lobular architecture. Nodules apparently
    were first described by Fitzhugh & Nelson (1947) who felt that they
    could be regarded as adenomas or as low grade hepatic cell carcinomas.
    The use of the second term is not clear because neither mitoses,
    tissue invasion, nor metastasis was observed. Although Ortega et al.
    (1956) reported small nodules in the livers of treated rats, and
    although they examined tissue loaned by Fitzhugh & Nelson's
    laboratory, they were entirely unimpressed by the lesions, referring
    to them as "focal incongruities".

        Almost 3 decades after the first study, there is no more agreement
    than is reflected in the preceding paragraph. The views of some
    pathologists remain diametrically opposed. This is true even though
    the finding of: (a) pulmonary metastases of hepatic cells in mice that
    had received DDT (Tomatis et al., 1972; Walker et al., 1973), ß-HCH,
    gamma-HCH, dieldrin, or phenobarbital (Thorpe & Walker, 1973); or (b)
    progression of liver enlargement beginning 12 weeks after cessation of
    ingestion of alpha-HCH by mice for 24 or 36 weeks (Nagasaki et al.,

    1974) or progressive increase in the size of liver nodules after DDT
    feeding was stopped (Tomatis et al., 1974b; Tomatis & Turusov, 1975);
    or even (c) in HCH-exposed mice the time-pattern of increase in liver
    weight (as reflected by body weight), which gained momentum only after
    a delay of 4 weeks but showed a further acceleration in the thirteenth
    week, in spite of decreased food consumption (Tomii et al., 1972),
    would appear to establish, without question, that at least some of the
    liver changes produced by these compounds in rodents are malignant.

        Of course, the reason for disagreement is that the tumours
    produced by DDT, other organochlorine insecticides, and phenobarbital
    differ in their biochemistry and are not malignant in the classical
    sense. Specifically, (a) they do not actively invade tissues; (b)
    their "metastases" do not grow; (c) they produce little shortening of
    life span; and (d) mice receiving 5.5 (mg/kg)/day as a result of
    dietary intake of DDT show a decrease in the success of
    transplantation and a significant increase in survival in mice in
    which tumours grew following inoculation with an otherwise uniformly
    transplantable and uniformly fatal ependymoma (Laws, 1971).

        Although the displacement of liver cells to the lung occasionally
    seen after prolonged dosage with DDT is usually referred to as
    metastasis, it might better be called embolism because the lesion does
    not progress and, therefore, lacks the clinical significance of a real
    metastasis. Because it does not grow, the lesion is hard to find. A
    number of investigators have failed to mention liver cells in the
    spleen, lymph nodes, or lungs, and some have stated specifically that
    they were not found (Nagasaki, 1973; Rossi et al., 1977).

        Perhaps the most illuminating study of the liver changes caused by
    DDT is that of Kuwabara & Takayama (1974). They used fluorinyl
    acetamide (2,7-FAA) as a positive control in their studies of DDT and
    HCH. The 3 compounds were given at dietary concentrations of 250, 250,
    and 600 mg/kg, respectively. The lesion caused by 2,7-FAA differed
    from those caused by either of the other compounds in 3 ways: (a) it
    started as hyper-plastic nodules rather than as isolated cell changes;
    (b) the final lesion was hepatocellular carcinoma in contrast with the
    adenoma caused by DDT or HCH; and (c) alpha-fetoprotein was formed,
    which did not occur with DDT or HCH. Other workers have also failed to
    find alpha-fetoprotein in mice treated with an organochlorine
    insecticide (Hanada et al., 1973).

        It must be emphasized that the organochlorine insecticides and
    phenobarbital do not produce, in other animals, the early, visible
    changes in the endoplasmic reticulum that are so characteristic of
    some rodents and that progress to tumour formation in rodents. That
    these compounds do not lead to tumour formation in other animals might
    have been predicted by the fact that they do not cause the early
    changes, characterized by hypertrophy, margination, and lipospheres.

        All available evidence indicates that man does not appear to be
    susceptible to the tumorigenic action of the organochlorine
    insecticides and phenobarbital. No increase in the occurrence of
    tumours has been found in heavily-exposed populations. This includes
    groups of workers who manufacture and formulate DDT and dieldrin and
    who have been examined carefully for tumours (Laws et al., 1967;
    Jager, 1970).

        Finally, a study based on a complete tumour registry did not
    indicate any increase of tumours attributable to phenobarbital among
    men and women who received heavy, essentially lifelong dosing with
    this drug for the control of epilepsy (Clemmesen et al., 1974).

        In summary, in spite of disagreement about the interpretation of
    the liver cell changes, there is general agreement about their
    development and appearance. The change that can be detected first and
    can be produced by the smallest effective dosage involves the
    endoplasmic reticulum. The initial change is reversible, but, even
    more important, it is peculiar to rodents. There is no evidence that
    anything from the first increase in endoplasmic reticulum to the final
    development of a highly nodular liver with occasional displacement of
    cells to the lung has any bearing on the health of man or other
    animals in which the endoplasmic reticulum does not respond in this
    way.

    7.1.10  Mutagenicity

        DDT has been tested in a number of ways for possible mutational
    effects. Shirasu et al. (1976) listed DDT as a negative chemical in
    microbial mutagenicity screening studies on 166 pesticides. The test
    system consisted of rec-assay using H 17 Rec+ and M 45 Rec-strains
    of  Bacillus subtilis and reversion assays without metabolic
    activation using auzotrophic strains of  Escherichia coli (WP 2) and
     Salmonella typhimurium (Ames series). Further studies by the same
    authors, with metabolic activation, failed to reveal mutagenicity of
    DDT (Shirasu et al., 1977). McCann et al. (1975) and McCann & Ames
    (1976), reported negative results on DDE in  Salmonella typhimurium
    testing with metabolic activation.

        At a dosage of 105 mg/kg, DDT did not produce any increase in
    dominant lethals in mice (Epstein & Shafner, 1968). Concentrations of
    10 mg/kg or higher produced chromosome breaks and exchange figures in
    a marsupial somatic cell line (Palmer et al., 1972). Saturated
    solutions produced chromosome breaks in the root tips of onion and
    other plants (Vaarama, 1947). A slight mutagenic effect in mammals has
    been reported by Markarian (1966). Deletions plus gaps were reported
    to be more common in the chromosomes of mice that had received DDT.

        An unconventional test for mutagenicity involved examination of
    explants of pulmonary tissue from embryonic mice whose dams had been
    fed dietary concentrations of DDT of 10 and 50 mg/kg. An increase in

    diffuse hyperplasia and focal proliferation was observed, but a
    dosage-response relationship was not clear. Some of the embryos were
    allowed to live and the experiment was repeated in subsequent
    generations. There was no continuing progression of the reported
    changes in succeeding generations (Sabad et al., 1972).

    7.1.11  Teratogenicity

        When  p,p'-DDT was administered to pregnant mice at a rate of
    1 mg/kg on days 10, 12, and 17 of gestation, it was not teratogenic
    but did alter the gonads and decrease the fertility of the young,
    especially the females (McLachlan & Dixon, 1972). A single dose at the
    rate of 25 mg/kg or repeated doses of 2.5 (mg/kg) day given during
    pregnancy may be embryotoxic but not teratogenic to mice (Schmidt,
    1973). The reason why one or a few doses during pregnancy may be
    embryotoxic although the same dosage is harmless, when administered
    during the entire reproductive period, is of theoretical but not
    practical importance.

        Teratogenic effects of DDT have not been seen in studies of
    reproduction including those for 2 generations in rats, 6 generations
    in mice, and 3 generations in dogs (see section 7.1.7).

    7.2  Acquisition of Tolerance to DDT

        Because DDT stimulates microsomal, mixed-function enzymes and the
    action of these enzymes on DDT is one of detoxication, it might be
    expected that some tolerance might develop. Such tolerance has been
    demonstrated in the case of dieldrin; rats that received this compound
    for 28 days at a rate of 2 mg/kg survived 25 additional days at a rate
    of 5 mg/kg, a dosage that killed 70% of previously untreated rats
    (Hutterer et al., 1968). Apparently the possibility of tolerance to
    DDT has not been explored.

    7.3  Factors Influencing DDT Toxicity

    7.3.1  Dosage effect

    7.3.1.1  Dosage-effect of DDT

        Table 5 summarizes the acute oral and dermal toxicity of DDT in
    common laboratory animals, and Table 18 summarizes the subcutaneous
    intravenous and intraperitoneal toxicity. Both tables are condensed
    from an earlier review (Hayes, 1959) that gives references and
    additional details. It may be concluded that dissolved DDT is absorbed
    through all portals. Absorption of DDT powder through the skin is
    negligible. It is frequently impossible to put enough DDT dust on the
    skin of animals to kill them, so that an LD50 value for this
    formulation cannot be determined by the dermal route. Although
    formulation is important in determining the toxicity of DDT by other
    routes, the difference is not so great as it is in connexion with skin

    exposure. DDT is about 4 times more toxic when given intravenously
    than when given orally, and about 40 times more toxic when given
    intravenously than when given dermally.

        Table 18.  Acute subcutaneous, intravenous, and intraperitoneal LD50 of DDT
               in common laboratory animalsa

    Species      Formulation                    Subcutaneous    Intravenous    Intraperitoneal
                                                (mg/kg)         (mg/kg)        (mg/kg)

    Rat          Water suspension or powder     >2000
                 Oil solution                   200-1500        47             80-200

    Mouse        Water suspension or powder     1000-1500
                 Oil solution                   300

    Guineapig    Water suspension or powder
                 Oil solution                   900                            150

    Rabbit       Water suspension or powder
                 Oil solution                   250->3200       30-41          <2100

    Cat          Water suspension or powder
                 Oil solution                   <650            32

    Dog          Water suspension or powder
                 Oil solution                                   68

    Monkey       Water suspension or powder
                 Oil solution                                   55

    a  From: Hayes (1959).

        In general, DDT appears to be more toxic as a solution in
    vegetable oil or animal fat than when given in some petroleum
    fraction. Petroleum may act as a laxative. The heavier fractions are
    never absorbed and DDT dissolved in such fractions has to be extracted
    from the solvent in order to show toxicity.

        In summary, DDT is a compound of moderately acute toxicity.
    Compared with other organochlorine insecticides of equal or greater
    toxicity, it is remarkable in being only slightly absorbed by the
    skin.

        The effects of repeated doses of DDT are summarized in Table 17.

        The 90-dose oral LD50 of technical DDT in rats is
    46.0 (mg/kg)/day (Gaines, 1969). The chronicity index is 5.4. Thus the
    compound has only a moderate tendency to cause cumulative effects, and
    this limited tendency is fully explained by the accumulation of DDT
    itself in tissues as a result of continuing intake. In fact, this
    accumulations, which is strictly dosage dependent, is detectable at
    all measurable levels of intake. The relationship in man is shown in
    Fig. 4 (p. 138).

        If storage is considered undesirable  per se, then DDT is without
    a no-injurious-effect level. However, the same may be said for all
    compounds that are absorbed, for the presence of all of them in the
    bodies of exposed organisms -- perhaps at very low levels -- may be
    assumed; failure to demonstrate low levels of storage does not depend
    on physiology but only on the limitations of analytical chemistry and
    on the lack of persistence of chemists.

        A number of papers have reported no-effect levels for DDT within
    the variables investigated, namely: rat, 0.05 mg/kg (Lehman, 1965);
    dog, 8 mg/kg (Lehman, 1965); and monkey, 2.2-5.54 mg/kg (Durham et
    al., 1963).

        There remain reports of effects in animals at the lowest dosages
    investigated. For example, decreased serum albumin and increased ß-
    and gamma-globulins in the blood of rats and rabbits maintained on a
    dosage of 0.2 (mg/kg)/day for 3-11 months was reported by Kagan et al.
    (1969).

        In studies carried out in rats and dogs, toxicity, as measured by
    the maximum no-effect level, was seldom very different from the
    corresponding value resulting from 90 days of exposure at the same
    dosage range. The largest factor of difference observed when 33
    chemicals were investigated in rats was 12 (20 for minimal effect
    level), and for half of them the factor was 2 or less. In 21,
    rat-to-dog comparisons of long-term toxicity, in no instance was the
    dog more sensitive than the rat (Weil & McCollister, 1963). Although
    LD50 studies offer a poor basis for predicting long-term toxicity,
    the lowest dosage that will produce a minimum effect, when administered
    for the lifetime of rats, can be predicted with reasonable success
    from a test lasting only 7 days and with good success from a 90-day
    study (Weil et al., 1969). These results offer some perspective for
    judging the ultimate effect of exposure to compounds that have been
    commercially available for less than one human lifetime.

         Summary of long-term toxicity studies. The lowest dosages that
    have been studied in animals are of the same order of magnitude as
    those encountered by men who make or formulate DDT and, therefore,
    hundreds of times greater than the dosages encountered by the general

    population. The animal studies have been continued long after a steady
    state of storage has been achieved. From the results it can be
    concluded that bioaccumulation sufficient to produce neurotoxicity or
    other clinical effects, including a reduction of the life span, can
    occur only at dosage levels substantially higher than those
    encountered by the most heavily exposed workers. DDT dosages
    encountered by workers produced a small but detectable increase in
    liver changes (hypertrophy, margination, and liposphere formation) in
    some groups of mice and rats. The same changes occurred in low
    incidence in control mice and rats but not in other animals (see
    section 7.1.9).

    7.3.1.2  Dosage-effect of metabolites and o,p'-DDT

        Acute oral LD50 values of DDT metabolites commonly found in
    tissues of excreta are shown in Table 19. Readily absorbable
    formulations of the metabolites are less toxic than the most
    absorbable preparations of the parent compounds (see for example
    Table 5).

    Table 19.  Oral LD50 values of metabolites of DDT

    Compound   Species        LD50     Reference
                              (mg/kg)

    DDE        rat, male        880    Gaines, 1960
    DDE        rat, female     1240    Gaines, 1960
    ODE        mouse            700    von Oettingen & Sharpless, 1946
    DDE        mouse           1000    Domenjoz, 1946a,b
    TDE(DDD)   rat, male      >4000    Gaines, 1969
    DDA        rat             1900    Smith et al., 1946
    DDA        rat, male        740    Gaines, 1960
    DDA        rat, female      600    Gaines, 1960
    DDA        mouse            720    von Oettingen & Sharpless, 1946
    DDA        mouse            590    Domenjoz, 1946a,b

         DDA. Rats tolerate higher tissue levels of DDA than of DDT.
    Eighteen hours after intravenous injection of DDA at a rate of
    100 mg/kg, tissue levels were still higher than those usually found in
    animals, fatally poisoned by DDT (Judah, 1949).

        DDA produces less injury to the liver than DDT but produces
    greater damage to the kidney especially at high intravenous dosages
    (Lillie et al., 1947). This is consistent with the finding of Spicer
    et al. (1947) that, following administration of DDT, DDA constitutes a

    higher proportion of DDT-related compounds in the kidney (25%) than in
    any other tissue, e.g., 12% in the liver, 10% in the brain, and even
    less in other tissues.

         o,p'-DDT. At an oral dosage of 150 mg/kg,  p,p'-DDT produces
    severe illness in all rats and kills about half of them, but  o,p'-DDT
    at the same dosage does not produce illness even though the
    concentrations of the 2 compounds in the brain at various intervals
    after dosing are about the same. At a dosage of 3000 mg/kg,  o,p'-DDT
    produces mild to moderate illness, and the concentration in the brain
    is 5-9 times the concentration of  p,p'-DDT necessary to produce
    similar symptoms. Thus,  p,p'-DDT appears to be inherently more toxic
    than the  o,p'-isomer (Dale et al., 1966a).

    7.3.2  Age and sex

        Young animals eat more than adults in relationship to their body
    weight. For this and other reasons, they are often more susceptible
    than adults to poison in food. However, young animals are inherently
    less susceptible to certain compounds. There is no evidence that DDT
    is more toxic to the young than to the adults of any species,
    including man. In the rat, the young are less susceptible than adults
    to a single dose and about equally susceptible to repeated doses as
    shown in Table 20. According to Henderson & Woolley (1969), the
    relative insusceptibility of the young is associated with relatively
    poor absorption of DDT by the central nervous system and by the less
    inherent susceptibility of the young brain to DDT already absorbed by
    it. Further studies by the same authors (Henderson & Woolley, 1970)
    showed that fatal poisoning of both 10- and 60-day-old rats involved
    hyperexcitability and intense tremor followed by prostration and
    eventual respiratory failure. However, in the adult rat, DDT caused
    convulsions, an increase in respiration and heart rate, and a lethal
    increase in body temperature (40-42°C) prior to death, but the body
    temperature of the immature rat decreased during acute intoxication by
    DDT. The authors suggested that, whereas DDT is a direct depressant of
    respiration in both young and old rats, the additional toxic responses
    manifested by seizures and hyperthermia accounted for the increased
    lethality of DDT in mature animals.

        There is virtually no sex difference in the acute toxicity of DDT
    to rats; the LD50 was 113 and 118 in males and females, respectively
    (Gaines, 1960). When DDT is fed to rats at ordinary dietary levels,
    the 2 sexes store it equally. However, at higher dosages, females
    store more of the compound; the difference is explained mainly by the
    lower activity of the liver microsomal enzymes in female rats and,
    only in part, by the relatively higher food intake of the females.

    Table 20.  Effect of age on the toxicity of DDT to rats

    Number      Age           LD50        Reference
    of doses                  (mg/kg)a

       1        newborn         4000      Lu et al.. 1965
       1        newborn         2356      Harrison, 1975
       1        10 days          728      Henderson & Woolley, 1969
       1        14-16 days       437.8    Lu et al., 1965
       1        weanling         355.2    Lu et al,, 1965
       1        2 months         250      Henderson & Woolley, 1969
       1        3-4 months       194.5    Lu et al., 1965
       1        middle-aged      235.8    Lu et al,, 1965
       1        adult            225      Harrison, 1975
       4        preweaning       279.2    Lu et al., 1965
       4        adult            285.6    Lu et al., 1965

    a  Total intake one or more doses.

    7.3.3  Nutrition

         Fat. Nutrition influences the toxicity of DDT in connexion with
    both fat and protein. Fatness influences the amount that can be stored
    inactively in the body, and it is of importance in mitigating acute
    poisoning. This action of fatness or "good condition" has been noted
    in connexion with mammals (Spicer et al., 1947) and fish (Hoffmann &
    Surber, 1948). In contrast, laboratory animals are slightly more
    susceptible to repeated large doses administered as part of a diet
    containing a moderate proportion of fat (5% or more) than as part of a
    very low fat diet (0.5%) (Sauberlich & Baumann, 1947). This difference
    is thought to be associated with absorption from the gastrointestinal
    tract.

        Rats that have stored large amounts of DDT in the fat may suffer
    tremors, if starvation or some other cause leads to a mobilization of
    their fat (Fitzhugh & Nelson, 1947). If DDT intake is stopped when
    starvation begins, and, if the concentration of DDT is measured only
    once following the interval of starvation, the results may be erratic,
    reflecting, to a greater or lesser degree, both mobilization of fat
    and excretion of metabolites as in studies reported by Deichmann et
    al. (1972).

        However, in nature, starvation is more often partial than
    complete, and, if the original diet contains enough DDT to cause
    substantial storage, whatever food may be found in a period of
    scarcity is also likely to be contaminated. The initial effect of the

    mobilization of fat is to increase the concentration of DDT in the
    remaining fat and in other tissues. Excretion is increased in response
    to the increased tissue levels but may not be fast enough to prevent
    the accumulation of a toxic concentration in the brain. If intake of
    DDT is stopped, the increased rate of excretion eventually leads to
    reduced storage (Dale et al., 1962). These findings in rats have been
    confirmed, in regard to both the initial increase in the concentration
    of DDT (Dedek & Schmidt, 1972; Stenberg & Diky, 1973) and the later
    reduction (Brodeur & Lambert, 1973). Similar findings have been
    reported in birds (Adamczyk, 1971).

        The effect of fat mobilization on the toxicity of DDT is the same
    whether it is caused by withholding food or by disease that causes
    partial refusal of food (Hayes, 1975).

        It is highly unlikely that poisoning by DDT will be precipitated
    in man by starvation because: (a) very few subjects store the compound
    in concentrations as high as those required to demonstrate the
    phenomenon in rats; and (b) the metabolic rate in man is so much
    slower than that in rats that elimination of DDT in man would
    counterbalance that produced by its mobilization.

         Lipids. The association of lipids with the function of
    microsomal enzymes is generally recognized as is the fact that DDT
    induces these enzymes. Therefore, it might have been expected that DDT
    and essential fatty acids would interact. Tinsley & Lowry (1972) found
    that the growth of female rats receiving  p,p'-DDT at a dietary level
    of 150 mg/kg was depressed, if they received a diet deficient in
    essential fatty acids, but was slightly stimulated, if they received
    the same diet supplemented with these acids. Another variable
    influenced by the same factors was the ratio of various liver lipids.
    The changes in fatty acid composition were related to the
    proliferation of hepatic smooth and endoplasmic reticulum; it was
    suggested that DDT influenced essential fatty acid metabolism by
    increasing the demand for them.

        In contrast, a variety of diets (containing fats that may occur in
    the human diet and that were in approximately the same proportion as
    fats in the typical human food in the USA) had little or no influence
    on the storage of DDT and a wide range of pesticides fed to rats for 4
    generations in a combination of rates 200 times those found in the
    Market Basket Study of food in the USA (Adams et al., 1974).

         Ascorbic acid. In squirrel monkeys (and presumably in other
    species) only 2 days on an ascorbic acid-deficient diet impaired both
    the induction of  O-demethylase and the stimulation of the glucuronic
    acid system by DDT (5 mg/monkey/day) (Chadwick et al., 1971a). In
    guineapigs, maintenance of induction of microsomal enzymes required a
    higher dietary level of ascorbic acid than prevention of scurvy
    (Wagstaff, 1971).

         Protein. Smith & Stohlman (1945) found only slightly greater
    mortality and liver pathology in rats fed DDT at 500 mg/kg in a diet
    containing protein at 80 g/kg than in one containing 280 g/kg. The
    finding that low dietary protein predisposes to DDT poisoning has been
    confirmed (Sauberlich & Baumann, 1947; Boyd & DeCastro, 1968, 1970;
    Boyd & Krijnen, 1969); however, even zero protein intake increased
    toxicity only 4-fold, the smallest factor observed in comparable
    studies of 9 pesticides. The effect of protein deficiency on toxicity
    may involve a crippling of the microsomal enzymes of the liver or it
    may act synergistically with compounds that cause anorexia. Rats fed a
    diet containing casein at 810 g/kg exhibited evidence of renal
    overload and were more susceptible to DDT (Boyd & DeCastro, 1970).

    7.3.4  Species

        The acute toxicity and metabolism of DDT were studied in mice and
    hamsters because of the marked difference in their susceptibility to
    liver tumours induced by DDT. The central nervous systems of the 2
    species are equally sensitive, the concentration of DDT in their
    brains at death being similar. However, after an oral dosage of
    500 mg/kg, the DDT concentration in the mouse brain was twice that in
    the hamster. This cannot be explained by a difference in absorption,
    metabolism, or excretion but apparently is due to a difference, in the
    permeability of the blood/brain barrier in the 2 species. When animals
    receive DDT at a dietary level of 250 mg/kg for 6 weeks, the residues
    in fat and liver were 7/8 times higher in the mouse, a fact only
    partially explained by the greater food intake of the mouse relative
    to body weight. Although urinary excretion of 14C-DDT was similar in
    previously unexposed hamsters and mice, this excretion was stimulated
    in the hamster but little affected in the mouse by previous dietary
    exposure to DDT (Gingell & Wallcave, 1974).

        Mice also differ from rats in the hormonal regulation of the basic
    activity of hepatic microsomal mixed-function enzymes as well as in
    the response of these enzymes to inducers (Chhabra & Fouts, 1974).

    7.3.5  Other factors

        A number of other factors are known to influence the toxicity of
    some compounds, and the degree of difference may be very great in
    isolated instances. Factors that have been reviewed elsewhere (Hayes,
    1975) include (in addition to those listed above) interaction of
    compounds, strain, individual differences, isolation and crowding,
    other social and psychological factors, temperature, pressure and
    altitude, light and other radiation, circadian and other rhythms,
    seasonal differences, and relative humidity. None of these additional
    factors is known to have an important effect on the survival of
    animals receiving DDT. The possibility of the interaction of DDT with
    aldrin, pyrethrin, piperonyl butoxide, malathion, dichloropheno-
    xyacetic acid (2,4-D), and a number of food additives has been

    explored systematically without finding anything but simple additive
    results (Fitzhugh, 1966). However, pyrethrins, especially synergized
    pyrethrins, have an additive and perhaps synergistic effect on the
    changes in liver morphology associated with repeated doses of DDT in
    rats (Kimbrough et al., 1968).

    7.4  Human Studies

         Oral exposure. Table 21 summarizes the effects of one or a few
    carefully measured oral doses of DDT. The results are consistent with
    those in accidents reported by Garrett (1947) and Hsieh (1954) in
    which it was possible to estimate accurately the amount ingested. It
    may be concluded that a single dose at the rate of 10 mg/kg produced
    illness in some but not all subjects even though no vomiting occurred.
    In general, smaller doses did not produce illness, although a dosage
    of 6 mg/kg produced perspiration, headache, and nausea in a man who
    was sickly and who was hungry at the time of eating. Persons who were
    made sick by 10 mg/kg did not have convulsions, but convulsions
    occurred frequently when the dosage level was 16 mg/kg or greater
    (Hsieh, 1954). Rarely, a dosage as high as 20 mg/kg might be taken
    without apparent effect (MacCormack, 1945). Dosages at least as high
    as 285 mg/kg have been taken without fatal result (Garrett, 1947).
    However, large doses lead to prompt vomiting, so that the amount
    actually retained cannot be determined accurately.

        It has been noted, in the course of tests with volunteers, that
    dilute colloidal aqueous suspensions of DDT are odourless and
    tasteless (Domenjoz, 1946a; Hoffman & Lendle, 1948). Saturated
    alcoholic solutions of DDT have a weak aromatic taste or rather odour.
    Some people find these solutions slightly anaesthetic to the tongue
    (Hoffman & Lendle, 1948). The taste of DDT in vegetable oil is so
    slight that many persons could not identify capsules containing 0,
    3.5, and 35 mg of DDT when they were presented separately but could
    arrange them in proper order when one of each was available for
    comparison (Hayes, personal communication, 1977).

        The possible clinical effects of many repeated doses of DDT were
    first explored by Fennah (1945). Because of his interest in predicting
    the results of indiscriminate use, he expressed the exposures in terms
    of environmental levels rather than in dosage units. The exposures
    were clearly higher than those ordinarily encountered. In one test,
    lasting a total of 11.5 months, Fennah daily inhaled 100 mg of pure
    DDT and drank water dusted at the rate of 3240 mg/m2. Much of the
    inhaled dust must have been deposited in the upper respiratory tract
    and swallowed. Later, for one month, Fennah ate food all of which had
    been sprayed at the rate of 2160 mg/m2 after it had been served. No
    ill-effect of any kind was observed.

        Table 21.  Summary of the effects of one or a few oral doses of DDT on volunteers

    Dose (mg) and       Result                                              Reference
    formulation

    250 × 9,            No effect.                                          Domenjoz, 1946a
    suspension

    1500, butter        No effect, but lice killed when fed 6               MacCormack, 1945
    solution            and 12 h after dose.

    500, oil            No effect.                                          Neal et al., 1946
    solution

    700, oil            No effect.                                          Neal et al., 1946
    solution

    250, suspension     None except slight disturbance of                   Velbinger, 1947a,b
                        sensitivity of mouth.

    250, oil            Variable hyperesthesia of mouth,                    Velbinger, 1947a,b
    solution

    500, oil            Variable hyperesthesia of mouth.                    Velbinger, 1947a,b
    solution

    750, oil            Disturbance of sensitivity of lower part of         Velbinger, 1947a,b
    solution            face; uncertain gait; peak reaction (6 h after
                        ingestion) characterized by malaise, cold moist
                        skin, and hypersensitivity to contact;
                        reflexes normal.

    1000, oil           Same as above; no joint pains, fatigue, fear,       Velbinger, 1947a,b
    solution            or difficulty in seeing or hearing.

    1500, oil           Prickling of tongue and around mouth and nose       Velbinger, 1947a,b
    solution            beginning 2.5 h after dose; disturbance of
                        equilibrium; dizziness; confusion; tremor
                        of extremities; peak reaction (10 h after
                        ingestion) characterized by severe malaise,
                        headache, and fatigue; delayed vomiting;
                        almost complete recovery in 24 h.

        Some later studies on volunteers have been designed to explore the
    details of storage and excretion of DDT in man and to search for
    possible effects of dosages considered to be safe. In the first of
    these studies, men were given 0, 3.5, and 35 (mg/man)/day. These
    administered dosages, plus DDT measured in the men's food, resulted in
    dosage levels of 0.0021-0.0034, 0.038-0.063, and 0.36-0.61
    (mg/kg)/day, respectively, the exact value depending on the weight of
    each individual. Six volunteers received the highest dosage of
    technical DDT for 12 months, and 3 received it for 18 months. A
    smaller number of men ingested the lower dosage of technical DDT or
    one of the dosages of  p,p'-DDT for 12 or 18 months. No volunteer
    complained of any symptoms or showed, by the tests used, any sign of
    illness that did not have an easily recognizable cause clearly
    unrelated to the exposure to DDT. At intervals, the men were given a
    systems review, physical examination, and a variety of laboratory
    tests. Particular attention was given to the neurological examination
    and liver function tests, because the major effects of DDT in animals
    involve the nervous system and the liver (Hayes et al., 1956). The
    same result was obtained in a second study in which the same dosages
    were given for 21 months and the volunteers were observed for a
    minimum of 27 additional months (Hayes et al., 1971). Information on
    storage and excretion gathered in these studies has already been
    discussed in sections 6.2.1.1 and 6.3.1.1.

        Recently, DDT has been used on an experimental basis at dosage
    rates varying from 0.3 to 3 (mg/kg)/day for periods up to 7 months in
    an attempt to decrease serum bilirubin levels in selected patients
    with jaundice. No side-effects were observed. No improvement was noted
    in patients with jaundice based on cirrhosis who did not have any
    demonstrated liver enzymes deficiency. However, in a patient with
    familial, nonhaemolytic, unconjugated jaundice based on a deficienty
    of glucuronyl-transferase, treatment with DDT rapidly reduced the
    plasma bilirubin level to the normal range and relieved the patient of
    nausea and malaise from which he had suffered intermittently. The
    liver function tests as well as other laboratory findings remained
    normal. The improvement was maintained during the 6 months that DDT
    was administered, and had persisted for 7 additional months at the
    time the report was written. In this case, a dosage of 1.5 (mg/kg)/day
    produced a steady rise in plasma levels of  p,p'-DDT from an initial
    level of 0.005 mg/litre to a maximum of 1.33 mg/litre at the end of
    treatment. At this time, the concentration in body fat was 203 mg/kg.
    Plasma levels fell slowly after dosing was stopped (Thompson et al.,
    1969). The highest dally intake in this series was 6 times greater
    than the highest level administered in earlier studies of volunteers
    and about 7500 times greater than the DDT intake of the general
    population. The highest value for  p,p'-DDT in serum observed in the
    entire series was 1.330 mg/litre compared with 0.996 mg/litre, the
    highest value reported by Laws et al. (1967) for formulating plant
    workers.

         Dermal exposure. Depending on dosage, oral administration of DDT
    to volunteers either did not produce any illness or produced only
    brief poisoning similar to that seen in experimental animals. The oral
    dosage necessary to produce any clinical effect was almost always
    10 mg/kg or more. However, in 2 studies involving only 3 subjects in
    all, experimental dermal exposure to DDT was followed by fatigue,
    aching of the limbs, anxiety, or irritability, and other subjective
    complaints. Recovery was delayed for a month or more (Case, 1945;
    Wigglesworth, 1945). In neither study was there an independent
    control. Although the dosage was unmeasured, the amounts of DDT
    absorbed must have been much smaller than those involved in the oral
    tests. One of the studies involved self-experimentation by one man. A
    similar but somewhat more severe test on 6 volunteers did not produce
    any toxic or irritant effects at all (Dangerfield, 1946). In view of
    all other experiments and extensive practical experience, it must be
    concluded that the illnesses reported by Wigglesworth and by Case were
    unrelated to DDT.

        With the exceptions just mentioned, dermal exposure to DDT has not
    been associated with any illness or, usually, with any irritation
    (Wasicky & Unti, 1944; Draize et al., 1944; Cameron & Burgess, 1945;
    Fennah, 1945; Dangerfield, 1946; Chin & T'Ant, 1946; Domenjoz, 1946a;
    Haag et al., 1948). In fact, Hoffman & Lendle (1948) reported that
    even subcutaneous injection of colloidal suspensions of DDT in saline
    in concentrations up to 30 mg/litre did not cause irritation.
    Zein-el-Dine (1946) reported that DDT-impregnated clothing caused a
    slight, transient dermatitis, but the method of impregnation was not
    stated and the absence of solvent was not guaranteed. In other more
    thorough studies DDT-impregnated clothing was found to be non-
    irritating (Cameron & Burgess, 1945; Domenjoz, 1946a).

        Small pads impregnated with different formulations of DDT were
    applied to the inner surface of the forearm of 32 volunteers whose
    cutaneous sensation had previously been measured for a period of 5
    weeks. Pads impregnated with all the elements of the formulation
    except DDT were applied to the corresponding position of the other arm
    as a control. Powdered DDT and a solution of DDT at 50 g/litre showed
    little effect. Solutions in olive oil and petrolaturn at 100 g/litre
    and 200 g/litre did not show any remarkable effect on sensation of
    pain, cold, or heat but reduced tactile sensation in most cases so
    that the minimum pressure that could arouse the tactile sensation was
    1-2.5 g/cm2 higher than for the control (Chim & T'Ant, 1946).

         Respiratory exposure. Neal et al. (1944) reported almost
    continuous daily exposures to aerosols sufficient to leave a white
    deposit of DDT on the nasal vibrissae of the volunteers. This exposure
    produced moderate irritation of the nose, throat, and eyes. Except for
    this irritation during exposure, there were no symptoms, and

    laboratory tests and physical examination, including neurological
    evaluation, failed to reveal any significant changes. The studies by
    Fennah (1945), which involved both respiratory and oral exposure, did
    not produce any detectable ill-effects.

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

    8.1  Retrospective Studies on DDT-Exposed Populations

    8.1.1  Epidemiological surveillance of persons occupationally exposed
           to DDT

        The safety record of DDT is phenomenally good. It has been used
    for mass delousing in such a way that the bodies and inner clothing of
    thousands of people of all ages and states of health have been
    liberally dusted with the compound. By necessity, the persons applying
    the DDT work in a cloud of the material. Other subjects have sprayed
    the interior of hundreds of millions of homes in tropical and
    subtropical countries under conditions involving (Wolfe et al., 1959)
    extensive dermal and respiratory exposure. A smaller number of men
    have made or formulated DDT for many years. Extensive experience and
    numerous medical studies of groups of workers have been reviewed
    (Hayes, 1959). Dermatitis was commonly observed among men who used DDT
    solutions. The rashes were clearly due to the solvent, especially
    kerosene. As often happens with rashes caused by petroleum
    distillates, they were most severe in men when they first started work
    and cleared in a few days unless contamination was exceptionally
    severe. A smaller number of workers experienced mild narcotic effects
    (vertigo and nausea) from solvents when working in confined spaces.
    Gil & Miron (1949) reported that some persons suffered temporary
    irritability, fatigue, and other ill-defined symptoms after exposure
    to the dusty atmosphere of a delousing station, but the relation of
    these atypical findings to DDT was not clear. With these exceptions
    due largely to solvents, no illnesses clearly attributable to the
    formulations, much less to DDT, were revealed by the early studies.

        Ortelee (1958) carried out clinical and laboratory examinations of
    40 workers, all of whom were exposed to DDT and some of whom were
    exposed to a number of other pesticides. The men had been employed at
    this work with heavy exposure for 0.4 to 6.5 years and with slightly
    less exposure for as much as eight years. Exposure was so intense
    that, during working hours, many of the men were coated with a heavy
    layer of concentrated DDT dust. By comparing their excretion of DDA
    with that of volunteers given known doses of DDT, it was possible to
    estimate that the average dosages of 3 groups of the workers with
    different degrees of occupational exposure were 14, 30, and 42 mg/man
    per day, respectively. With the exception of the excretion of DDA and
    the occurrence of a few cases of minor irritation of the skin and
    eyes, no correlation was found between any abnormality and exposure to
    the insecticide. Since very large doses of DDT injure the nervous
    system and liver of experimental animals, special attention was given
    to a complete neurological examination and to laboratory tests for
    liver function. Although a few abnormalities were revealed, none was
    detected in relation to DDT.

        Thirty-five men employed from 11 to 19 years in a plant that had
    produced DDT continuously and exclusively since 1947 and, at the time
    of the study, was producing 2722 metric tonnes per month were studied
    by Laws et al. (1967). Findings from medical histories, physical
    examinations, routine clinical laboratory tests, and chest X-ray films
    did not reveal any ill-effects attributable to exposure to DDT. The
    overall range of storage of the sum of isomers and metabolites of DDT
    in the men's fat was 38-647 mg/kg compared with an average of 8 mg/kg
    for the general population. Based on their storage of DDT in fat and
    excretion of DDA in urine, it was estimated that the average daily
    intake of DDT by the 20 men with high occupational exposure was
    17.5-18 mg/man per day compared with an average of 0.028 mg/man per
    day for members of the general population. There was significant
    correlation ( r = +0.64) between the concentration of total DDT-related
    material in the fat and the serum of the workers. The average
    concentration in fat was 338 times higher than that in serum -- a
    factor about 3 times greater than that for people without occupational
    exposure. Compared to members of the general population, the workers
    were found to store a smaller proportion of DDT-related material in
    the form of DDE; the difference was shown to be related chiefly to
    intensity rather than to duration of exposure. DDE is relatively a
    much less important and DDA a much more important excretory product in
    occupationally-exposed men compared with men in the general
    population. A further study of the same men involved in DDT production
    is discussed in section 8.2.5.

        By far the largest number of heavily-exposed workers whose health
    has been investigated are those associated with malaria control in
    Brazil and India (WHO, 1973). In Brazil, periodic clinical
    examinations were made of 202 spraymen exposed to DDT for 6 or more
    years, 77 spraymen exposed for 13 years ending in 1959, and 406
    controls. In the first examination carried out in 1971, minor
    differences between exposed and unexposed groups were observed in some
    neurological tests, but this result was not confirmed by the second
    examination in the same year nor in subsequent examinations. During a
    3-year period, a survey of illnesses requiring medical care during the
    6 months preceding each periodic medical examination failed to
    demonstrate any differences between exposed and control groups. A
    relatively small number of analyses indicated that the concentration
    of DDT in the blood of spraymen was about three times higher than that
    of controls.

        In India, the blood levels of 144 spraymen were 7.5-15 times
    higher than those of the controls and were at least as high as those
    reported for workers who make and formulate DDT elsewhere (see
    Table 7). When the spraymen were examined, the only differences from
    the controls were that knee reflexes were brisker, slight tremor was
    more often present, and a timed Romberg test was more poorly performed
    by the spraymen. The positive results led to the selection of 20 men
    for re-examination by a neurologist who concluded that the differences

    found initially were not real or that the tests had returned to normal
    within the few months between the 2 examinations. The signs were not
    dosage-related, since they were not correlated with serum levels of
    DDT.

        It has been known for several years that substantial doses of DDT
    and several other organochlorine insecticides stimulate the microsomal
    enzymes of the liver. This property of DDT was put to practical use in
    treating a patient with familial, nonhaemolytic, unconjugated
    jaundice, as described earlier. It was, therefore, entirely expected
    that persons with sufficient occupational exposure to a variety of
    pesticides would be able to metabolize a test drug (phenazone) more
    rapidly on the average than persons without occupational exposure were
    able to do. However, the change was not one of significantly
    increasing the fastest normal rate but of bringing all the workers up
    to a high level. There was no indication that the change had any
    effect on the workers' health (Laws et al., 1967, 1973; Kolmodin et
    al., 1969; Poland et al., 1970).

        In addition to the studies already mentioned regarding workers
    with extensive storage, and excretion of DDT as a result of heavy
    exposure to DDT, studies have also been made of a larger number of
    workers with lesser storage and excretion following lesser exposure to
    DDT but greater exposure to other insecticides. Further studies (Long
    et al., 1969; Morgan & Roan, 1969, 1974; Warnick & Carter, 1972;
    Sandifer et al., 1972; Embry et al., 1972; Tsutsui et al., 1974; Ouw &
    Shandar, 1974) have failed to reveal effects of clinical significance
    among workers with prolonged, moderate exposure not only to
    organochlorine but also to organophosphorus and other types of
    insecticides. Small but statistically significant differences have
    appeared in the medical history or clinical laboratory results of some
    of these workers compared with the controls, but in no instance have
    the differences been of any medical importance, and dosage-response
    relationships have been unclear or absent. In several instances, the
    statistically significant differences have been opposite in different
    groups of workers; for example, creatinine phosphokinase activity was
    lower than that of controls in subjects applying the insecticide but
    higher in operators. Seasonal variations present one year were lacking
    the next. The possibility of adaptive change (other than enzyme
    induction) has been suggested (Tocci et al., 1969), but this, like the
    reality of the changes, remains unproved.

        In some instances statistically significant differences have been
    found between workers and controls selected from the general
    population in connexion with parameters that have no known biochemical
    relationship to DDT and for which another explanation has not been
    excluded. For example, Keil et al., (1972) reported significant linear
    correlations between serum vitamin A and plasma DDT, TDE, and DDE
    levels.

        There are a few reports of acute illness among workers attributed
    to exposure to mixtures of DDT and other materials. In so far as the
    dosage was very large, as in certain accidents that have occurred to
    individuals or groups in the general populations (see section 8.2.2),
    one would expect similar results. However, in at least one instance,
    headache, dizziness, nausea, vomiting, pain and numbness of the limbs,
    and general weakness beginning 1-1.5 h after entering a treated field
    (Kolyada & Mikhal'Chenkova, 1973) suggested food poisoning or
    hysteria.

        Finally, there are studies of workers exposed to DDT and various
    other pesticides that are reported to have produced a variety of
    subjective and even objective medical findings. Interpretation of
    these reports is difficult because: (a) the findings do not resemble
    those of poisoned animals or of persons poisoned as a result of
    accident or suicide; and (b) the papers fail to report how the medical
    findings and the absenteeism of the pesticide workers compares with
    those of workers of comparable age, sex, and exertion who are not
    exposed to chemicals. The fact that the workers in question were
    exposed to mixtures of pesticides is not in itself an explanation
    because studies on many workers who were exposed to mixtures have not
    revealed any consistent differences between exposed subjects and
    unexposed controls. However, an explanation may lie in the degree of
    exposure. Reports of very high levels of organochlorine compounds in
    blood samples and of DDT in milk samples from populations in which
    illness was found are discussed in sections 6.2.1.2 and 6.3.1.2.

        The reports under discussion tend to fall in 2 categories, those
    involving general debility and those involving a single organ or
    system. Conditions representative of general debility include
    dermatitis, subtle blood changes, general weakness, palpitations,
    functional angiospasm, headache, dizziness, diminished appetite,
    vomiting, lower abdominal pain, chronic gastritis, benign chronic
    hepatitis, isomnia, a sympathetic vascular/asthenic syndrome,
    vegetative dystonia, and confusion (Kostiuk & Mukhtrova, 1970; Bezugli
    et al., 1973).

        Organs, systems, or functions that have been studied with the
    exclusion of other organs, systems, or functions of the same workers
    include: the respiratory system (Boiko & Krasniuk, 1969), liver
    (Bezuglyi & Kaskevich, 1969), stomach (Krasniuk & Platonova, 1969;
    Platonova, 1970), kidneys (Krasniuk et al., 1968), adrenals (Bakseyev,
    1973), skin (Karimov, 1969, 1970), and labour and the puerperium
    (Komarova, 1970; Nikitina, 1974). An indication that the difficulties
    under discussion are not serious is their reversal or prophylaxis by
    means of diet. Lescenko & Polonskaia (1969) described in detail two
    dietary supplements composed of ordinary foods plus sea-kale and a
    selection of vitamins and trace metals. Organochlorine-exposed workers
    who received these diet products showed a normalization of protein
    metabolism manifested by an increase in total serum protein, improved

    lipid metabolism, and enriched vitamin and trace element supplies in
    the organism. All of these effects led to an improvement in the
    detoxifying function of the liver, which was viewed as the most
    frequent site of adverse effects of exposure to organochlorine
    compounds.

    8.1.2  Epidemiology of DDT poisoning in the general population:
           accidents and suicides

        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. The facts were reviewed in
    sections 6.2.13 and 6.3.1.2. Briefly, DDT and some of its derivatives
    are found in all or nearly all persons in the population. The
    concentration is higher in tissues that have a high neutral fat
    content. Thus, for members of the general public the concentration of
    DDT-related compounds in adipose tissue is 100 or more times greater
    than the concentration in plasma (Laws et al., 1967). However, in
    spite of this great difference, sufficiently sensitive methods have
    demonstrated DDT in all tissues including the fetus and in all body
    fluids including human milk. These relationships are exactly what
    would be predicted from what is known of the storage of drugs and
    other compounds. Actual chemical demonstration of the distribution of
    DDT has been established for several years. Thus, its occurrence was
    first reported in human tissue (Howell, 1948), in tissue of the
    general population (Laug et al., 1951) in human milk (Laug et al.,
    1951), and in the human fetus (Denés, 1962).

        There is extensive evidence that the mount of DDT and related
    material in the general diet in the USA has decreased as the use of
    DDT in that country has decreased, especially its use on forage.
    During the early 1950s, total DDT-related intake was approximately
    0.265 mg/man per day and that for DDT was 0.163 mg/man per day (Walker
    et al., 1954). The average intake of DDT-related compounds based on a
    very large number of samples collected in different parts of the
    country during 1964-67 was 0.063 mg/man per day and that for DDT was
    0.028 mg/man per day (Duggan, 1968). With decreased use of DDT, a
    gradual decrease in the storage of DDT and related material in human
    fat would be expected. Because only a few samples of fat were
    collected in the early studies of human tissue, there is some
    statistical uncertainty as to whether the decrease in storage that has
    been observed is real or whether it merely reflects variation due to
    sampling. In any event, by 1968, the average storage level of total
    DDT-equivalent material in fat was 7.67 mg/kg and that for DDT was
    1.46 mg/kg. These averages were based on just over 3000 samples
    collected during the first half of 1968. The number of samples
    involved in this particular study was greater than the sum of all of
    the samples used in early studies. The best available values for
    concentrations in serum are 0.0294 mg/litre for total DDT-equivalent
    and 0.0047 mg/litre for  p,p'-DDT.

        Cases of accidental and suicidal poisoning in which the effects
    were clearly caused by DDT are summarized in Table 22. All of these
    cases involved ingestion. The signs and symptoms of poisoning were
    entirely consistent with those observed in volunteers, except that the
    spectrum of effects was broader because some of the accidental and
    suicidal doses were very high. A few persons have apparently been
    killed by uncomplicated DDT poisoning, but none of these cases was
    reported in detail. Death has been caused much more frequently by the
    ingestion of solutions of DDT, but in most instances the signs and
    symptoms were predominantly or exclusively those of poisoning by the
    solvent (Hayes, 1959). This does not mean that the toxicity of the
    solvent always predominates. For example, the recurrent convulsions in
    a case reported by Cunningham & Hill (1952), though more
    characteristic of poisoning by one of the cyclodienes, was certainly
    not typical of solvent poisoning. A 2-year-old child drank an unknown
    quantity of fly spray of which 5% was DDT, but the nature of the other
    active ingredients or the solvent was unknown. About 1 h after taking
    the material, the child became unconscious and had a generalized,
    sustained convulsion. Convulsions were present when the child was
    hospitalized 2 h after taking the poison, but the fits were controlled
    by barbiturates and other sedatives. Convulsions reoccurred on the
    fourth day and again on the twenty-first day but ceased each time
    following renewal of treatment. On the twelfth day, it was noted that
    the patient was deaf. Hearing began to improve about the twenty-fourth
    day and was normal, as were other neurological and psychic findings,
    when the patient was seen about 2.5 months after the accident.

        Clinical effects of one toxicant may be modified by combining it
    with another. For example, prolonged illness would not be expected
    from ingestion of DDT at a rate of 27 mg/kg. However, when DDT and
    lindane were ingested in a suicidal attempt at dosages thought to be
    27 mg/kg and 18 mg/kg, respectively, clinical remission of convulsions
    and liver involvement was delayed until the twentieth day, and the EEG
    did not return to normal until the thirty-ninth day (Eskenasy, 1972).

        There have not been any accidents of suicided involving raspatory
    or dermal exposure to DDT leading to recognized signs and symptoms of
    poisoning, even though sufficient respiratory exposure to aerosols or
    sufficient dermal exposure to solutions can cause poisoning in
    animals; the difference is certainly one of dosage.

        It has been alleged that DDT causes or contributes to a wide
    variety of diseases of man and animals not previously recognized as
    being associated with any chemical. Such diseases include
    cardiovascular disease, cancer, atypical pneumonia, retrolental
    fibroplasia, poliomyelitis, hepatitis, and "neuropsychiatric
    manifestations" (Biskind & Beiber, 1949; Biskind, 1952, 1953; and
    others). Without exception the causes of these diseases were unknown
    or at least unproved at the time of the allegation. Needless to say,

        Table 22.  Summary of the effects of the accidental or suicidal ingestion of DDT

    Individual dose       Results and reference
    (mg) Formulation
    Number of persons

    300-4500              Onset in 1 h; vomiting; restlessness; headache; heart weak and
    in food               slow; recovery next day (Mulhens, 1946).
    1 man

    unknown dose          Onset in 2-2.5 h; all subjects weak and giddy; 4 subjects vomited;
    in tarts              2 subjects hospitalized; one subject confused, uncoordinated, weak;
    25 man                one subject with palpitations and numbness of hands; recovery in
                          24-48 h (Mackerras & West, 1946).

    5000-6000             Onset 2-3 h; throbbing headache; dizziness; incoordination;
    in pancakes           paraesthesia of extremities; urge to defaecate; wide nonreacting
    3 men                 pupils; reduced vision; dysarthria; facial weakness; tremor; ataxic
                          gait; reduced sensitivity to touch; reduced reflexes; positive Romberg;
                          slightly low blood pressure and persistent irregular heart action;
                          partial recovery in 2-3 days, but slight jaundice appeared 4-5 days
                          after ingestion and lasted 3-4 days; all subjects normal 19 days after
                          poisoning except for irregular heart action in one subject (Naevested,
                          1947).

    2000                  No illness (Naevested, 1947).
    in pancakes
    2 men

    up to 20 000          Onset in 30-60 min in those most severely affected; men first
    in bread              seen 2-3 h after ingestion; in spite of severe early vomiting that
    28 men                reduced the effective dose, severity of illness and especially intensity of
                          numbness and paralysis of extremities proportional to amount of DDT
                          ingested; all but 8 men recovered in 48 h; 5 others fully recovered in 2
                          weeks, but 3 men still had some weakness and ataxia of the hands
                          5 weeks after ingestion (Garrett, 1947, 1950, unpublished data).

    unknown dose          Onset about 3.5 h after ingestion; total of about 85 cases of which 37
    in flour              were hospitalized; symptoms mild and similar to those in earlier
    about 100 women       outbreaks except for gastrointestinal disturbance in most severe cases
                          including abdominal pain and diarrhoea as well as nausea; most
                          subjects fully recovered in 24 h (Jude & Girard, 1949).

    unknown dose          Symptoms in established cases similar to those reported earlier
    14 cases              (Francone et al., 1952).

    Table 22 (Cont'd)

    Individual dose       Results and reference
    (mg) Formulation
    Number of persons

    286-1716              With the exception of one man who was already sick when he received
    in meatballs          a dosage of 6 mg/kg, poisoning did not occur at dosages of
    8 cases, 11           5.1-10.3 mg/kg. Ingestion of 16.3-120.5 mg/kg produced excessive
    exposed               perspiration, nausea, vomiting, convulsions, headache, increased
                          salivation, tremors, tachycardia, and cyanosis of the lips. Onset
                          varied from 2-6 h depending on dosage. Recovery required as much
                          as 2 days (Hsieh, 1954).

    unknown dose          Death 13 h after suicidal ingestion (Committee on Pesticides, 1951).
    1 case

    unknown dose          Twenty-two separate cases, including 15 attempted suicides; some
    22 unrelated cases    complicated by solvents; 3 deaths (Committee on Pesticides, 1951).

    the charge that DDT predisposes to poliomyelitis was dropped after the
    disease was controlled through the use of vaccines. Unfortunately,
    there is no immediate possibility of controlling cardiovascular
    disease, cancer, or many of the less common conditions in man that
    have been ascribed to DDT. In the meantime, such irresponsible claims
    could produce great harm and, if taken seriously, even interfere with
    the scientific search for true causes and realistic means of
    preventing the conditions in question.

    8.1.3  Epidemiology of DDT poisoning in infants and young children

        Nothing is known fundamentally to distinguish the epidemiology of
    DDT poisoning among children from that among adults. In both
    instances, poisoning has never been confirmed, except where the dosage
    was large, usually as the result of an accident and usually involving
    gross carelessness. Probably a larger number of cases have occurred in
    infants simply because they are more likely to eat and drink
    formulations that they find in unlabelled containers frequently
    originally intended for food. However, as far as DDT is concerned, all
    the large outbreaks of poisoning have involved adults under military
    conditions, thus children were not exposed. As might be expected, some
    deaths of adults but none of children has apparently involved suicide.
    Most, if not all, cases in adults were uncomplicated poisoning by DDT,
    but several cases in children involved the drinking of solutions so
    that the signs and symptoms were actually caused by the solvent
    (Reingold & Lasky, 1947).

    8.2  Clinical and Epidemiological Studies of the Effects of DDT on
         Specific Organs and Systems

    8.2.1  Haemopoietic system and immunology

        In acute poisoning, a slight decrease in haemaglobin and a
    moderate leukocytosis without any constant deviation in the
    differential white count have been observed in volunteers (Velbinger,
    1947a,b). These findings are considered secondary to the neurological
    effects.

        There is a strong tendency to blame blood dyscrasias, other
    manifestations of "hypersensitivity", and, in fact, many diseases of
    unknown cause on any new chemical that gains widespread attention. DDT
    was no exception. A review of the early literature (Hayes, 1959)
    indicates that blood dyscrasias and an unbelievable range of other
    diseases were, in fact, blamed on DDT. Only a circumstantial
    relationship was ever established between these diseases and exposure
    to DDT, and this is true of the small number of reports of blood
    dyscrasias (Murray et al., 1973) or angioneurotic oedema (Vanat &
    Vanat, 1971) that have appeared recently. Later, fewer new reports
    appeared linking DDT to diseases of unknown cause, although the use of
    DDT increased greatly. It is true that available tests do not make it
    possible to exclude a particular compound as a cause of an isolated
    case of blood dyscrasia. However, it is noteworthy that the rate at
    which these disorders occur has remained essentially unchanged since
    before DDT was introduced (Hayes, 1975).

    8.2.2  Nervous system

        The effects of carefully measured doses of DDT that proved to be
    just above the minimum toxic level are best described from studies of
    volunteers (see section 7.4). Similar early signs and symptoms have
    been encountered in cases of accidental poisoning that frequently
    progressed to more severe illness as described in section 8.1.2.

        Briefly, the earliest symptom of poisoning by DDT is
    hyperaesthesia of the mouth and lower part of the face. This is
    followed by paraesthesia of the same area and of the tongue and then
    by dizziness, an objective disturbance of equilibrium, paraesthesia
    and tremor of the extremities, confusion, malaise, headache, fatigue,
    and delayed vomiting. The vomiting is probably of central origin and
    not due to local irritation. Convulsions occur only in severe
    poisoning.

        Onset may be as soon as 30 min after ingestion of a large dose or
    as late as 6 h after smaller but still toxic doses. Recovery from mild
    poisoning is essentially complete in 24 h, but recovery from severe
    poisoning requires several days. In two instances, there was some
    residual weakness and ataxia of the hands, 5 weeks after ingestion.

        Electroencephalograms were obtained from 73 workers exposed to
    DDT, HCH, and chlorobenzilate for periods ranging from 7 months to 20
    years. Just over 78% of the records were normal and 21.9% were
    abnormal. The most severe changes involved persons exposed to the 3
    compounds for 1-2 years; less severe changes were seen with either
    shorter or longer exposure. The changes were not correlated with age,
    the range and mean of age for those judged abnormal being almost
    identical to these values for persons considered normal. Some of the
    records showed bitemporal sharp waves with shifting lateralization
    combined with low voltage theta activity. Other records showed spike
    complexes, paroxymal discharges composed of slow and sharp waves most
    pronounced anteriorly, and low voltage rhythmic spikes posteriorly.
    None of the persons examined showed any abnormal clinical neurological
    finding (Israeli & Mayersdorf, 1973; Mayersdorf & Israeli, 1974). The
    incidence of abnormal electroencephalograms in the general population
    is 9.0% or 9.2%, according to other investigators cited by Israeli &
    Mayersdorf. Czegledi-Janko & Avar (1970) considered that nonspecific
    EEG abnormalities occurred in 10-20% of the general population.

        The frequency and degree of olfactory disorders, especially in the
    ability to detect peppermint and acetic acid in an olfactory analyser,
    were reported to be greater among persons exposed to pesticides, and
    to increase with duration of exposure (Salihodzaev & Ferstat, 1972).
    Whether any of the persons exposed to pesticides experienced any
    clinical difficulty or social inconvenience associated with olfactory
    sensation is not clear.

    8.2.3  Renal system

        There is no indication of renal damage in people, accidentally
    poisoned by DDT, or in workers heavily exposed to it.

    8.2.4  Gastrointestinal system

        Except for vomiting, which probably is of central origin, the
    gastrointestinal system has not been affected in acute poisoning.

    8.2.5  Liver

        Involvement of the liver has been mentioned in only a small
    proportion of cases of accidental poisoning by DDT. In 3 men who ate
    pancakes made with DDT and thus ingested 5000-6000 mg each, slight
    jaundice appeared after 4-5 days and lasted 3-4 days (Naevested,
    1947). Hepatic involvement and convulsions were reported in an
    unsuccessful suicide attempt by ingesting DDT and lindane (Eskenasy,
    1972).

        Laws et al. (1973) made a detailed study of the liver function of
    31 men who had made and formulated DDT and who had been the subjects
    of an earlier study (see section 8.1.1). Judging from their excretion

    and storage, the men's exposure was equivalent to oral intakes of DDT
    at rates ranging from 3.6 to 18 mg/man per day for periods ranging
    from 16 to 25 years and averaging 21 years. All tests were in the
    normal range for total protein, albumin, total bilirubin, thymol
    turbidity, and retention of sulfobromophthalein sodium (BSP). One man
    had mild elevations in levels of both alkaline phosphatase
    (EC 3.1.3.1) (16 units) and serum glutamic pyruvic transaminase
    (EC 2.6.1.2) SGPT (42 units). Another man had an alkaline phosphatase
    concentration of 14 units, while a third man had an SGPT level of 49
    units. The alpha-fetoprotein test was negative for all 20 of the men
    tested.

    8.2.5.1  Liver enzymes

        The induction of human microsomal enzymes of the liver by various
    drugs was well known when Kolmodin et al. (1969) demonstrated this
    effect in workers exposed to a variety of pesticides, including DDT.
    Later, Poland et al. (1970) showed that workers who made and
    formulated DDT and absorbed it at an average rate of about
    0.25 (mg/kg)/day metabolized phenylbutazone more rapidly on the
    average than controls and excreted more 6ß-hydroxycortisol.
    Occupational exposure increased the drug-metabolizing ability of some
    workers, so that they all metabolized test drugs with the efficiency
    of those members of the general population who were most efficient in
    this respect. The concentration of  p,p'-DDT in the serum of the
    workers studied by Poland averaged 0.573 mg/litre. In other workers
    with less exposure to DDT, as indicated by average serum levels of
    0.052 mg/litre, there was no increase in the urinary excretion of
    D-glucaric acid, which is increased by a number of exogenous and
    endogenous substances that induce microsomal enzymes (Morgan & Roan,
    1974).

        Thompson et al. (1969) demonstrated, in a different way, the
    induction of microsomal enzymes by using DDT at a dosage of
    1.5 (mg/kg)/day for 6 months in the successful treatment of
    unconjugated hyperbilirubinaemia. In a similar way, Rappolt (1970)
    used DDT to promote metabolism of an overdose of phenobarbital. It is
    of interest that the levels of DDE in the serum of some workers
    studied by Morgan & Roan (1974) approached those of workers studied by
    Poland et al. (1970). The lack of induction in one group and its
    presence in the other suggests that enzymes are induced in man more
    readily by DDT than by DDE.

        DDT promotes its own metabolism in some species of laboratory
    animals. That the same is true in man is indicated by the fact that
    storage of DDT is relatively less at higher dosages (see Fig. 4).
    However, the metabolism and subsequent excretion of DDT can be
    promoted even more by other inducing agents. Patients who received
    phenobarbital or, more especially, phenytoin stored much less DDT than
    other persons with similar exposure to DDT (Davies et al., 1969a;

    Edmundson et al., 1970b; Watson et al., 1972). This result concerning
    phenytoin was confirmed by McQueen et al. (1972) who also showed that
    other drugs produced a smaller but still highly significant reduction
    in DDT storage. Establishment of a reduced equilibrium appeared to
    require about 2 months. Within this period, the regression of the
    level of DDT plus DDE on duration of treatment with phenytoin was
    highly significant ( P < 0.001).

        At the end of 9 months' treatment, the body fat of nonepileptic
    volunteers given phenytoin at a rate of 300 mg/man per day contained
    an average of 25% of the DDT and 39% of the DDE concentrations
    originally present before administration of the drug (Davies et al.,
    1971).

        The same was true of workers whose exposure was greater than that
    of the general population. Maintenance doses of phenobarbital,
    phenytoin, or a combination of the two kept the storage levels of
    several organochlorine insecticides in epileptic workers as low as, or
    lower than levels in the general population (Schoor, 1970; Kwalick,
    1971).

    8.2.5.2  Other biochemical observations

        A positive linear correlation has been reported for the
    concentrations of vitamin A and of DDT-related compounds in the serum
    of men with at least 5 years of occupational exposure to DDT. However,
    the workers' DDT levels were little higher than those of persons in
    the general population (see Table 7), and their vitamin A levels were
    within normal limits (Keil et al., 1972). Perhaps they were better fed
    than the controls.

        Compared to 86 unexposed workers, the serum total cholesterol
    values of 206 workers in a chemical plant where unidentified
    organochlorine insecticides were made and formulated were higher in
    workers who were less than 25 years old, lower in those between 25-34
    years and 35-44 years, and higher in those who were 45 years old or
    more. The differences were significant only for the oldest groups
    (Wassermann et al., 1970a).

    8.2.6  Cardiovascular system

        The small amount of knowledge concerning the effect of DDT on the
    human heart fails to show whether cardiac arrhythmia might be a
    possible cause of death in acute poisoning, as is true in some species
    of laboratory animals. Palpitations, tachycardia, and irregular heart
    action have been noted in some, but not all cases of acute poisoning
    (Mackerras & West, 1946; Naevested, 1947; Hsieh, 1954).

    8.2.7  Reproduction

        There is no indication that DDT has influenced reproduction except
    to increase it as an indirect result of disease control, especially
    malaria control.

        After Laws et al. (1967) had completed their study, Wilcox (1967)
    found that the 36 most heavily exposed workers involved had fathered
    58 children before they began working at the DDT factory and 93
    children afterwards.

        O'Leary et al. (1970c) did not find any significant relationship
    between abortion and blood levels of DDT-related compounds.

    8.2.8  Endocrine organs

        Average protein-bound iodine (PBI)levels of 0.0542 and
    0.0693 mg/litre, respectively, were reported in the sera of 42 workers
    occupationally-exposed to organochlorine insecticides and in 51
    workers who were not exposed. The difference was statistically
    significant even though all values fell within the normal range of
    0.04-0.08 mg/litre (Wassermann, D. et al., 1971). It was not recorded
    whether the workers involved were from the same factory as those with
    10 or more years of occupational exposure whose plasma DDT levels were
    reported by Wassermann et al. (1970c) (see Table 7). The small
    difference in PBI levels is difficult to evaluate. It was the view of
    Clifford & Weil (1972) that there was not any evidence that
    occupational exposure had had an effect on human endocrine organs.

         TDE. Following the demonstration (discussed in section 7.1.8)
    that TDE caused atrophy of a part of the adrenal cortex of dogs,
     o,p'-TDE, and to a lesser degree  m,p'-TDE, have been used in man,
    under the name of mitotane, in the hope of controlling excessive
    cortical secretion or of reducing the size of adrenal tumors. The
    underlying condition may be hyperplasia or adrenocortical carcinoma.
    The dosage given has varied from 7 to 285 (mg/kg)/day, but a dosage of
    approximately 100 (mg/kg)/day for many weeks has been necessary to
    produce any benefit in man (Bergenstal et al., 1960; Wallace et al.,
    1961; Gallagher et al., 1962; Verdon et al., 1962; Bledsoe et al.,
    1964; and Southern et al., 1966a,b).

        The effects of idiopathic hyperplasia may be controlled; in fact a
    state of adrenal insufficiency may be produced (Canlorbe et al., 1971;
    Sizonenko et al., 1974).

         o,p'-TDE may also give symptomatic relief of excessive
    adrenocortical activity secondary to a tumour that produces ACTH
    (Carey et al., 1973).

        A favourable response was produced in about one-fourth to one-half
    of patients with inoperable adrenocortical carcinoma (Canlorbe et al.,
    1971; Hoffman & Mattox, 1972; Lubitz et al., 1973; Montgomery &
    Struck, 1973). In fact, an occasional cure, involving complete
    regression of metastases, was produced by chemotherapy including
     o,p'-TDE (Perevodchikova et al., 1972; Schick, 1973). More commonly,
    symptoms were relieved and life was prolonged by little more than 7-8
    months (Canlorbe et al., 1971; Hoffman & Mattox, 1972; Lubitz et al.,
    1973). The success of treatment was often indicated early on by a
    reduction in steroid excretion (Hoffman & Mattox, 1972; Lubitz et al.,
    1973).

        The large dosage of  o,p'-TDE necessary to produce clinical
    benefit often produced general lassitude, anorexia, nausea, vomiting,
    diarrhoea, and dermatitis (Naruse et al., 1970; Hoffman & Mattox,
    1972; Nitshke & Link, 1972; Perevodcikova et al., 1972; Lubitz et al.,
    1973). Apathy ranged from mild dulling of interest to profound
    psychotic depression (Hoffman & Mattox, 1972). More rarely,
    gynaecomastia, haematuria, leukopenia, and thrombocytopenia have been
    reported (Luton et al., 1972; Perevodcikova et al., 1972). The
    symptoms disappeared soon after administration of the drug ceased or
    when the dosage was reduced (Perevodcikova et al., 1972).

        Even large, therapeutic doses of  o,p'-TDE did not cause
    histological alterations in the adrenals in man (Wallace et al.,
    1961). Furthermore, dosages in the therapeutic range (specifically
    those between 110 and 140 (mg/kg)/day did not produce any detectable
    injury to the liver, kidney, or bone marrow. All patients treated in
    this way experienced significant anorexia and nausea, and some showed
    central nervous system depression varying from lethargy to somnolence.
    These toxic effects cleared when dosing was discontinued (Bergenstal
    et al., 1960).

        Kupfer (1967) reviewed extensive literature that indicated that
    the effect in man and other species, except the dog, is caused by
    stimulation of corticoid metabolism by massive doses of  o,p'-TDE and
    not by any direct effect on the adrenal. Southern et al. (1966a,b)
    agreed that the effect was predominantly extra-adrenal in man, when
    the drug was first given, but offered evidence that adrenal secretion
    of cortisol was eventually reduced. However, even if therapeutic doses
    eventually have a direct effect on the adrenal, doses encountered by
    workers exposed to technical DDT do not (Clifford & Weil, 1972; Morgan
    & Roan, 1973).

    8.2.9  Carcinogenicity

        Laws et al. (1967) did not find any case of cancer or blood
    dyscrasia among the 35 heavily exposed workers in a DDT factory nor
    did the medical records of 63 men who had worked there for more than 5
    years reveal these diseases. Two men were employed who had a history

    of successfully treated cancer before they came to work, but no
    employee had contracted cancer during the 19 years that the plant had
    been in operation; during this period, the work force varied from 111
    to 135.

        In the USA, the total death rates for cancer of the liver and its
    biliary passages (classified individually as "primary", "secondary",
    and "not stated whether primary or secondary") lead to the conclusion
    that there has been a significant, almost constant decrease in the
    total rate of liver cancer deaths from 8.8 in 1930 to 8.4 in 1944
    (when DDT was introduced) to 5.6 in 1972. This almost constant decline
    in total liver cancer death rates for the past 42 years offers no
    evidence of any increase in liver cancer deaths since the introduction
    of the first organochlorine pesticide into the environment. The
    decrease in liver cancer deaths is even more significant in light of
    the increasing life span of the general population in the USA, which
    has resulted in an increased percentage of the population at risk from
    cancer over these years. In spite of the limitations inherent in the
    interpretation of such data, this record is a reminder that, more than
    30 years after the introduction of DDT, there is no evidence,
    whatsoever, that DDT is carcinogenic in man (Deichmann & MacDonald,
    1976, 1977).

        In the USA, the incidence of cancer is lower in rural counties
    than in metropolitan areas in general (Mason et al., 1975).

        It is sometimes implied that epidemiological evidence is useless
    for revealing the carcinogenicity of a material for man unless it
    involves large numbers of people who have been exposed to the material
    for most or all of a lifetime. The fact is that some human carcinogens
    have been detected through their occurrence in high incidence in small
    groups for periods of much less than 25 years. What was commonly
    considered the first recognition of chemical carcinogenesis in man
    depended on the observations made by a single surgeon (Pott, 1775) in
    a small fraction of his patients. Such was the intensity of the
    exposure of the apprentices of chimney sweepers that cancer of the
    scrotum often appeared at puberty. The editor responsible for
    compiling the writings of Pott (1790) added a footnote indicating that
    he had seen such a cancer in "an infant under eight years of age". It
    must be understood that boys did not usually become apprentice chimney
    sweepers before they were four-years-old. In connection with tumours
    of the bladder mainly caused by ß-naphthylamine but to a lesser degree
    by other aromatic amines, Hueper (1942) reviewed cases in which the
    time from the first exposure to recognition of symptoms was 8-41,
    9-28, and 2-35 years; in one series of 83 cases, 71% of the tumours
    appeared from 1 to 15 years after exposure. The same author cited
    reports (p. 104) of cases of malignant epitheliomas in persons exposed
    to pitch for 18, 24, 24, and 36 months, respectively. Kleinfeld (1967)
    reported 50-76% incidence of bladder cancer among several groups of

    workers. He also noted a sharp drop in incidence of this condition
    following decrease -- but not discontinuation -- of occupational
    exposure to ß-naphthylamine.

    8.2.10  Mutagenicity

        Evidence regarding the mutagenic activity of DDT and its
    significance in man is uncertain partly because the chromosomal
    changes that are examined are sensitive to viral infections and
    chemotherapy. The latter may not be recognized at the time of sampling
    and may not have been shown to injure health through a mutagenic
    mechanism.

        Comparing samples collected in winter and during the peak season
    of pesticide application, a slight increase in chromatid breaks was
    reported in the cultured lymphocytes of workers exposed to a wide
    variety of insecticides said to include DDT, although this was claimed
    at a time when the use of DDT was banned. A somewhat larger increase
    was reported for men exposed mainly to herbicides (Yoder et al.,
    1973). In another study, lymphocytes cultured from workers with an
    average DDT plasma level of 0.999 mg/litre showed significantly more
    chromosomal and chromatid aberrations than cells cultured from
    controls with an average plasma level of 0.275 mg/litre. The
    difference was not significant in other comparisons in which the
    average plasma levels were 1.030 versus 0.380 mg/litre and 0.240
    versus 0.030 mg/litre, respectively (Rabello et al., 1975).
    Examination of all of the data presented by the authors suggests a
    simple dosage-effect relationship was present, with a detectable
    effect starting somewhere between 0.2 and 0.4 mg/litre and increasing
    at levels higher than 0.4 mg/litre.

    8.3  Factors Influencing DDT Toxicity

        There is no evidence that any factor except dosage is of practical
    importance in determining DDT toxicity in man. Factors that have been
    considered as possibly affecting asymptomatic storage of DDT include
    age, sex, and race. Differences observed in connexion with these
    factors are small, medically insignificant, and probably secondary to
    dosage (Hayes, 1975).

        Storage has also been reported to be greater in the tissues of
    people with certain diseases (Deichmann & Radomski, 1968; Raclomski et
    al., 1968; Vas'kovskaja, 1969; Dacre & Jennings, 1970; Jonczyk et al.,
    1974). Again, the reported differences are small, and the highest
    values for the samples in question are small compared with those found
    in healthy workers (Hayes, 1975). Furthermore, a number of authors
    have reported a similar range of storage in persons undergoing minor,
    elective surgery and in those who have died from various causes (Hayes
    et al., 1958; Dale et al., 1965; Robinson et al., 1965; Wassermann et
    al., 1965). Some authors (Hunter et al., 1963; Robinson et al., 1965;

    Hoffman et al., 1967; Hoffman, 1968; Morgan & Roan, 1970) have failed
    to find any relationship between storage of insecticides and the cause
    of death. Where a relationship was found, there was often the
    possibility that the higher values were found in diseases that
    involved some degree of wasting prior to death. Casarett et al. (1968)
    found that higher values occurred in persons who had 3 characteristics
    in common: emaciation, cancer, and widespread abnormality of the
    liver.

        A slightly greater storage of DDT and DDE that was reported in
    persons who underwent splenectomy for hepatosplenic schistosomiasis
    compared with those operated on for other conditions, mainly hernia
    was statistically significant. No such difference was observed in
    connexion with dieldrin, ß-HCH, or heptachlor epoxide (Wassermann et
    al., 1975). Whereas it was speculated that the increased storage of
    DDT and DDE might have been the result of a reduction in metabolism,
    secondary to liver injury, the possibility of greater exposure as a
    result of greater use of DDT in irrigated areas was not excluded.

    8.4  Treatment of Poisoning in Man

        No useful guidance regarding treatment has been obtained from the
    very few cases of DDT poisoning that have occurred. Animal studies
    indicate that sedatives, ionic calcium, and glucose or another ready
    source of energy would be useful. On the basis of experience in
    treating people poisoned by different convulsive poisons, it seems
    likely that diazepam would be beneficial.

    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

        Food represents the major source of intake of DDT in the general
    population. It has been estimated (section 5.1.5) that over 90% of the
    DDT stored in the general population is derived from food. Around
    1965, when the use of DDT was at its peak, intake in the USA was
    approximately 0.04 mg/man per day from food, less than 0.000046 mg/man
    per day from water, less than 0.00006 mg/man per day from urban air
    and less than 0.0005 mg/man per day from the air in small agricultural
    communities. The reason for the qualification "less than", is that the
    intakes were calculated from the highest concentrations reported in
    drinking-water and air.

        Although total intake of DDT from food has not been measured in
    some parts of the world, worldwide measurements of the storage of DDT
    and its metabolites in human body fat indicate that the extremes of
    total exposure have varied by a factor of about 10, but that total
    exposure for most populations has varied by a factor of no more than 3
    (see Table 9).

        DDT in the dust in a house (as indicated either by a history of
    extensive household application of insecticides or by the finding of
    relatively high levels of DDT in house dust) can contribute to the
    storage of DDT-related compounds in persons living in the house
    (section 5.1.4). However, although the contribution of house dust to
    DDT intake has been established, it is not quite clear how this
    contribution occurs. Some of the dust may contaminate food in the
    process of preparation and some may be inhaled and later swallowed
    after deposition in the upper airway. It is difficult to believe that
    enough DDT is present in such houses in the form of vapour or
    respirable dust to cause a substantial increase in the total exposure
    of the inhabitants, but no critical study has been made of this
    matter. Clearly, some DDT will enter by the respiratory route.

    9.1.2  Infants and children

        At birth, infants tend to have slightly lower levels of DDT than
    adults in the same population. This is because the placenta offers
    partial protection against the passage of DDT and related compounds.

        Although human milk tends to have a somewhat higher concentration
    of DDT than cow's milk (see section 6.3.1.3), the difference, if any,
    that this makes to the rate at which breast-fed and bottle-fed babies
    store the compound has not been established. It is possible that the
    conditional acceptable daily intake (ADI) might be exceeded in an

    infant wholly fed on breast milk. However, the ADI is calculated on
    the basis of lifetime exposure, and short-term variations can be
    regarded as not having any significance.

        The only really important way in which the exposure of infants and
    children differs from that of adults in the same community involves
    accidental exposure (see section 8.1.3).

    9.1.3  Occupational groups

        Occupational exposure (section 5.3) to DDT is initially almost
    exclusively through the respiratory and dermal routes. However, the
    particles of many insecticidal dusts, wettable powders, and sprays are
    too large to reach the lower respiratory tract. As a result, most of
    the particles inhaled are deposited in the upper respiratory tract,
    carried to the pharynx by ciliary action, and eventually swallowed
    (section 6.1.1).

        Although dermal exposure to DDT is high under some occupational
    situations, the effect is minimal because the compound is so poorly
    absorbed through the skin (section 6.1.3). The excellent safety record
    of DDT, never matched by any other insecticide used in antimalaria
    campaigns, other vector control programmes, and agriculture, is based
    mainly on its poor absorption through the skin.

        The number of people with full-time occupational exposure to DDT
    alone is small. For example, at the time of one study, the only
    factory making the compound in the USA produced 2722 metric tonnes per
    month using a work force of about 145. Following the recommendations
    of an Expert Committee, the World Health Organization studied spraymen
    who had applied only DDT for 5 years or more. Only 272 suitable
    subjects could be located in Brazil and only 144 in India. The
    concentrations of DDT and its derivatives in the blood of the
    preliminary and main study groups in India were 0.761 and
    1.272 mg/litre, respectively. The blood levels of spraymen in Brazil
    were about 3 times those of the controls.

        The absorbed dosage of the men who made and formulated DDT for 10
    years or more was about 18 mg/man per day (see section 8.1.1). The
    exposure of other workers, notably those applying DDT for agricultural
    purposes has usually been an order of magnitude less (see Table 7).

    9.2  Effects of Exposure

        No adverse effects have been described at repeated dosages of
    1.5 (mg/kg)/day or less (see section 7.4). The large number of
    measurements that have been made on samples from human populations
    have not made it possible to define a maximum dosage that man can
    absorb without any adverse effect but have highlighted the finding
    that the high levels found in volunteers and workers were harmless for
    at least 25 years.

        Table 23 summarizes the clinical aspects of DDT in man.

    Table 23.  Dosage-effects of DDT in man

    Single dose
    (mg/kg)             Observation

    Unknown             Fatal
    16-286              Prompt vomiting at higher doses
                        (all poisoned, convulsions in some)
     6-10               Moderate poisoning

    Repeated
    exposure
    (mg/kg)/day

    1.5                 Administered as therapy for 6 monthsa
    0.5                 Administered to volunteers for 21 monthsa
    0.5                 Exposure of workers for 6.5 yearsa
    0.25                Exposure of workers for 25 yearsa
    0.0025              Intake of population in the USA, 1953-54a
    0.0002              Intake of population in the USA, 1969-70a

    a Without any adverse effect.

        In considering the safety of workers who are employed in the DDT
    industry, it is useful to consider the results of animal experiments.
    Rats withstand a daily dosage at least 10 times that of these workers
    without any detectable clinical effect (section 7, Table 17), although
    minimal reversible tissue changes may be present. Dogs and monkeys
    also withstand a daily dosage 10 times higher than that of the
    workers, but they do not show the tissue changes, which seem to be
    peculiar to some rodents. Because workers have tolerated high dosages
    of DDT for over a fifth of a lifetime without detectable harm and
    since animals withstand larger dosages for an entire lifetime without
    injury, there is good reason to predict the continuing safety of the
    workers.

        The experience already gained from workers can be used to predict
    the future safety of the general population in relation to DDT. Many
    workers have now been exposed to DDT for much more than one-fifth of
    their life span. Since they have not suffered detectable harm, it
    seems most unlikely that the general population will be harmed by
    dosages 200-1250 times smaller than those to which the workers are
    exposed. It has been shown for at least 2 animal species that toxicity
    resulting from a lifetime of exposure is seldom very different from

    toxicity resulting from 90 days of exposure at the same dosage rate.
    The largest factor of difference observed when 33 chemicals were
    investigated was 20, and, for half of them, the factor was 2 or less
    (see section 7.3.1.1). Ninety days constitute about one-eighth of the
    lifespan of a rat, and this is less than the fraction of the human
    life span that has been studied so far.

    9.3  Carcinogenicity and Mutagenicity

        Liver tumours are produced in mice by many chlorinated compounds,
    including DDT. There is also evidence to suggest that DDT metabolites
    DDE and TDE (DDD) produce hepatic tumours in mice and that TDE also
    produces lung tumours. Information on the tumorigenicity of DDT in
    rats (see section 7.1.9) is conflicting; some studies report tumour
    formation while other studies report negative data. Carcinogenicity
    studies in the hamster were negative (see section 7.1.9). The
    occurrence of tumours in some rodents only, casts doubt on the
    significance of the phenomenon and on extrapolation of the findings to
    man.

        Studies on the incidence of all cancers reported in those parts of
    some countries with known high agricultural use of DDT in the 1950s
    and early 1960s have not demonstrated any trends in any type of cancer
    associated with the use of DDT in relation to these areas (see section
    8.2.9).

        The question as to whether DDT is carcinogenic in man has not been
    answered unequivocally. Although the cross-sectional epidemiological
    studies on workers exposed to DDT and the observation studies on
    volunteers are limited, there is not any currently available evidence
    to suggest that DDT is tumorigenic or carcinogenic in man (see section
    8.2.9).

        Recent studies on  in vitro bacterial test systems with or
    without metabolic activation have not shown any evidence that either
    DDT or DDE is mutagenic (see section 8.2.10). The evidence for the
    mutagenicity of DDT in mammalian test systems is inconclusive.

    9.4  Effects on Microsomal Enzymes

        There is no doubt that exposure to DDT results in the induction of
    microsomal mixed function oxidases and causes marked morphological
    changes in the liver of some rodents (see section 7.1.9). In some
    rodents, notably the mouse, these morphological changes have been
    related to tumorigenicity. Microsomal enzymes are also induced by DDT
    in other species, but the liver does not show the same morphological
    changes.

        The only effect for which something approaching a threshold has
    been demonstrated is the induction of microsomal enzymes in workers in

    association with an average serum value of 0.573 mg/litre for  p,p'-
    DDT but not in workers with serum levels as high as 0.052 mg/litre, a
    value essentially identical to the highest reported for the general
    population. Furthermore, although some groups of workers experienced
    an increase in their average enzyme activity, no person exceeded the
    range of activity found in normal people in the general population.

        DDT will not induce liver microsomal enzymes in the general
    population because their intake of the compound is so much less than
    the smallest dosage capable of producing this effect in animals or man
    (see sections 7.1.4.2 and 8.2.5.1).

    9.5  Reproduction and Teratogenicity

        Effects on reproduction in mammals have been studied in the mouse,
    the rat, and the dog (see section 7.1.7). In the mouse, a multi-
    generation study at dietary levels of DDT of 25, 100, and 250 mg/kg
    showed effects on fertility and reproduction only at the highest
    level, equivalent to 33 (mg/kg)/day. In the rat, normal reproduction
    was maintained at a dietary level of 200 mg/kg. In the dog, dietary
    intake at dosages up to 10 (mg/kg)/day did not produce any effects on
    reproduction other than earlier estrus in the DDT-treated females.

        In man, there is no indication that DDT affects reproduction (see
    section 8.2.7); no impairment of fertility was observed in a study of
    men occupationally-exposed for more than 10 years to a measured
    average daily intake in the region of 18 mg/man per day (equivalent to
    0.25 (mg/kg)/day).

        Studies in the mouse, the rat, and the dog have not shown any
    evidence of teratogenicity. In the mouse, dosage at the rate of
    1 mg/kg was not teratogenic; a single dosage of 25 mg/kg or repeated
    doses at the rate of 2.5 mg/kg were embrytoxic but not teratogenic.

    9.6  Immunosuppression

        DDT appears to have a depressant effect on the immune system
    although the evidence is by no means conclusive. Rats and rabbits
    receiving DDT in aqueous suspension at a concentration of 200 mg/litre
    showed a depression in antibody formation and decrease in at least one
    globulin fraction of the blood. Rats receiving a dosage of
    0.25 (mg/kg)/day by gavage did not show any changes in the phagocytic
    activity of the white blood cells. In the guineapig, dosages of
    1-20 mg/kg did not have any effects on antitoxin production but
    produced a reduction in tissue histamine levels (see section 7.1.1).

    9.7  Nutritional Effects and Other Factors

        Animal studies indicate that nutritional status influences the
    toxicity of DDT (see section 7.3.3). The preferential storage of DDT
    in fat can mitigate the effect of acute poisoning. If rats that have

    stored large amounts of DDT are starved, they may suffer toxic effects
    due to mobilization of fat and DDT.

        In man, nutritional status will have a similar effect to that
    found in other 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
    and the lower metabolic rate of man results in slower mobilization. In
    fact, severe weight loss sometimes does cause some increase in storage
    of DDT in connexion with certain wasting diseases; however, people
    with full-time occupational exposure to DDT average 10 times more
    storage than the highest values reported in connection with disease
    but do not exhibit predisposition to the diseases in question (see
    section 6.2.1.3).

        Although young animals are often more susceptible to toxic
    chemicals than adults, there is no evidence that DDT is more toxic to
    young animals of any species including man. In fact, in the rat, the
    young are less susceptible to a single dose than the adults (see
    section 7.3.2, Table 20).

    9.8  Dosage-Effect Relationships

        Dosage-effect relationships for DDT in man have been observed in
    connection with acute poisoning (see Table 23), excretion, and storage
    (see Fig. 4), and the induction of microsomal enzymes, which 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 that causes an increase in
    tumours in male mice of a susceptible strain but not in females of any
    strain (see section 7.1.9). As shown in Table 24, this dosage in
    workers is less than the no-effect levels for rats, dogs, and monkeys
    and far less than the dosage at which rats, mice, and dogs
    successfully reproduce for generations. The equilibrium levels of DDT
    and its metabolites found in the blood and fat of people with
    full-time occupational exposure and the much lower levels found in the
    general population have not been associated with any adverse effects.

    9.9  Recommendations on Levels of Exposure

        The data from intake, exposure, and levels in populations supports
    the current conditional ADI for DDT, which affords a considerable
    margin of safety.

        If the total intake of DDT from food and other sources rises above
    0.005 (mg/kg)/day (the conditional ADI) then the situation should be
    investigated.

        Table 24.  Dosage-effect of DDT in animals

    Single         Route            Observation
    (mg/kg)

    3000           dermal           LD50 of powder in adult rat
    2356-4000      oral             LD50 of oil solution in newborn rat
    250-500        dermal           LD50 of oil solution in adult rat
    250            oral             LD50 of oil solution in adult rat

    Repeated
    Exposure
    (mg/kg)/day

    300            subcutaneous     inhibition of testicular growth in cockerels
    41-80          oral             increased mortality in rats, 2-year study
    41-80          oral             100% mortality in dogs in 39-49 months
    41-80          oral             100% mortality in monkeys, 70 days
    21-40          oral             25% mortality in dogs in 39-49 months
    33.2           oral             harmful to reproduction in mice
    13.3           oral             slight reduction in lactation and survival of some but not all
                                    generations of mice (6 generation test)
    10             oral             no harmful effect on reproduction in dog (3 generation test)
    10             oral             no harmful effect on reproduction in rat (2 generation test)
    5-10           oral             no-effect level in dog, 2 generations
    2.6-5          oral             no-effect level in monkey, 7.5 years
    0.63-1.25      oral             no-effect level in rat, 2 year test
    0.16-0.31      oral             risk of liver tumours doubled in male mice but no effect in
                                    female
    0.3            oral             no-effect level for induction of microsomal enzymes in rats

        The concentration of DDT in the air in industrial, agricultural,
    or disease control areas should not exceed 1 mg/m3 on a time-
    weighted basis (40 h per week). Several countries have their own
    standards that range from 0.1 to 1 mg/m3, which seem to afford an
    acceptable margin of safety.

        There is ample reason to predict the continuing safety of workers
    producing and using DDT. No harmful effect has ever been reported in
    vector control operators who have applied DDT during the last 3
    decades in public health programmes. Nevertheless, as in the case of
    any chemical, occupational safety and health measures should always be
    applied to ensure that contact with DDT by workers is kept to a
    minimum.

        The only index of exposure of DDT or its metabolites is the
    analysis of these compounds in tissues or excreta. For most purposes
    it is best to sample serum or plasma. In subjects with relatively
    constant, prolonged exposure, concentrations of DDT and its
    metabolites in the blood are in equilibrium with those in all other
    tissues, including adipose tissue. In subjects who have accidentally
    received a single large dose, the concentration in the brain is
    reflected more accurately by a serum sample than by a fat sample.

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    ANNEX

    TRANSFORMATION OF P,P'-DDT IN THE ENVIRONMENTa

    1  Abiotic Transformations

        Since 1969, the photolysis of  p,p'-DDT (Annex Fig. 1, formula
    II) and of its known primary environmental degradation product, DDE
    (1,1-dichloro-2,2-bis [ p-chlorophenyl]ethylene; Annex Fig. 1,
    formula VII) has been studied by irradiation in methanol at 260 nm
    (Plimmer et al., 1970). The products formed were DDMU (1-chloro-2,2-
    bis [ p-chlorophenyl] ethylene; Annex Fig. 1, Formula III),
    dichlorobenzophenone (Annex Fig 1, formula IV) and dichlorobiphenyl
    (Annex Fig. 1, Formula V). The formation of the last compounds
    proceeded via dichlorobenzophenone as an intermediate. The detection
    of the chlorinated biphenyl raised the question as to whether DDT
    might be a source of PCBs in the environment. Upon investigation of
    the reaction pathways leading to these substances, it was shown that
    DDE is converted to 3,6-dichlorofluoroenone (Annex Fig. 1, formula VI;
    Plimmer & Klingebiel, 1969) which is photooxidized to 3,3'-dichloro-
    biphenyl-2-carboxylic acid. Subsequent decarboxylation of this acid
    could yield traces of 3,3'-dichlorobiphenyl; the decarbonylation of
    another photolysis product of DDT, trichlorobenzophenone, could yield
    traces of trichlorobiphenyl, demonstrating that the formation of PCBs
    with more than 2 chlorine atoms was also possible (Plimmer &
    Klingebiel, 1973).

        Irradiation studies with substances in organic solvents are not
    necessarily predictive for the environment. However, studies with DDT
    vapour in sunlight confirmed the results obtained with dissolved
    substances. The proposed pathways of DDT photolysis under
    environmental conditions is shown in Annex Fig. 1 (Moilanen & Crosby,
    1973).

        In 1972, DDE was irradiated in solvents, in the solid state, and
    in the gaseous phase, with UV-light of various wavelengths. The
    results are shown in Annex Fig. 2. Besides the known photoproducts IV
    and III, a "trichlorinated DDMU" (Annex Fig. 2, formula VIII) and 2
    compounds with longer side chains (Annex Fig 2, formula IX and X) were
    identified; these 2 substances, however, were formed only upon
    irradiation in a solvent and originated from the reaction with the
    solvent (Kerner et al., 1972).

    a  Prepared by Dr F. Korte at the request of the Task Group.

    

        In a recent study on the photoisomerization and photodegradation
    of DDE under simulated natural conditions (inert solvents, a good
    hydrogen donor solvent, UV-light similar or equal 300 nm), the
    compound VIII (Annex Fig. 2) with the 3 phenyl-bound chlorine atoms
    was detected and characterized as a mixture of the E- and Z-isomers
    which were separated and isolated. Both isomers were also found in
    natural samples like tobacco and pine needles. DDMU was also detected
    in these studies and 2 substances so far unknown, a tetrachlorinated
    phenanthrene and a tetra-ring-chlorinated diphenylethylene; the
    formation of tri- and tetrachlorobiphenyls was confirmed (Göthe et
    al., 1976).

        The behaviour of compounds in their adsorbed form is equally as
    significant environmentally as their photochemical behaviour in the
    gaseous and solid states.

        Irradiation of DDE, adsorbed on silicagel, with wavelengths
    > 230 nm, resulted in the formation of dichlorobenzophenone and its
    trichlorinated analogue. Irradiation of DDT and DDE in the solid form
    in an oxygen stream with wavelengths > 230 nm, resulted in partial
    mineralization to give carbon dioxide and hydrochloric acid (Gab et
    al., 1975).

        The results presented here show that a large number of DDT-derived
    chlorinated compounds must be included, when considering the possible
    effects of DDT residues in the ecosphere.

    2  Biotransformations Other Than Mammalian Metabolism

    2.1  Birds

        Two main pathways of DDT metabolism exist in mammals i.e.,
    dehydrochlorination to DDE (Annex Fig. 3, formula VII) and stepwise
    degradation to DDA (bis-[ p-chlorophenyl] acetic acid) via TDE (DDD)
    (1,1-dichloro-2,2-bis [ p-chlorophenyl] ethane; Annex Fig. 3,
    formula I). However, in birds, the pathway varies with species, and
    data from studies on the administration of chronic and acute dosages
    of DDT to pigeons, quail, and blackbirds show that DDE is the primary
    metabolic product in the first 2 species, and TDE (DDD) in the third
    (Bailey et al., 1972). The TDE-path-way does exist in the pigeon as a
    minor pathway but, in contrast to mammals, only as far as DDMU (Annex
    Fig. 3, formula III).

        Thus, DDA, the degradation product of DDMU excreted by mammals, is
    not formed in the pigeon (Bunyan et al., 1966; Bailey et al., 1969).
    When its precursors in mammals, DDMS (1-chloro-2,2-bis[ p-
    chlorophenyl] ethane; Annex Fig. 3, formula XI) and DDN U (1,1-bis
    [ p-chlorophenyl] ethylene; Annex Fig. 3, formula XII) are
    administered to the pigeon, they are rapidly converted: DDMS is
    converted to DDMU, and DDNU is metabolized quickly and excreted as
    DDNS (1,1-bis [ p-chlorophenyl] ethane; Annex Fig. 3, formula XIII),
    a metabolite that was not found in mammals (Bailey et al., 1972). The
    metabolic pathways for DDT in the pigeon are shown in Annex Fig. 3.

    2.2  Insects

        Investigations on the detoxication mechanism of DDT in insects are
    interesting as regards the problem of resistance. In general, the
    phenomenon of insect resistance is related to detoxication of the
    insecticide by metabolization to nontoxic compounds. The metabolic
    pathways of DDT in insects are many and depend on species and even on
    strains.

        Annex Fig. 4 shows only the major metabolic products.

        The first conversion product identified in resistant houseflies
    was DDE. This conversion is catalyzed by the enzyme DDT-dehydro-
    chlorinase (EC 4.5.1.1) which had already been isolated in the pure
    form in the 1950s. Further insect metabolites of DDT are DDD (isolated
    for example from  Stomoxys calcitrans), DDA (isolated from example
    from  Quiscalus quiscula, Heliothis virescens and  Coleomegilla
     maculata) and dichlorobenzophenone (from  Leucophae). The
    detoxiation of DDT in  Triatoma infestans, Drosophila melanogaster,
     Culex tarsalis, and other species is performed by hydroxylation and
    results in kelthane (Annex Fig. 4, formula XV), a substance which is a
    commercial acaricide. A great number of unidentified and water-soluble
    conversion products of DDT was observed in many species as reviewed by
    Klein & Korte (1970).

    

        Although TDE (DDD) has not been found as a DDT-metabolite in
     Culex tarsalis, it has been concluded from differences between
    susceptible and resistant strains that it is an intermediate in the
    further degradation of DDT. After application of TDE 14C to this
    insect, DDMU and DDDOH (1,1-bis[ p-chlorophenyl]-2,2-dichloroethanol;
    Annex Fig. 5, formula XVI) were found as major metabolites;
    furthermore, 3 polar compounds were chromatographically identical with
    DDA, DBH (dichlorobenzhydrole; Annex Fig. 5, formula XVIII) and PCBA
    ( p-chlorobenzoic acid; Annex Fig. 5, formula XVIII), which were also
    observed after application of DDMU-14C (Plapp et al., 1965). The
    occurrence of PCBA indicates the complete breakdown of one of the 2
    rings of DDT and thus the possibility of a complete biological
    degradation of the whole molecule.

    2.3  Higher plants

        Although the transformation of DDT in higher plants is rather
    limited (2% in spinach within 18 days, 5% in cabbage within 14 weeks),
    it must not be neglected since a considerable part of the DDT used on
    a worldwide basis is applied, intentionally or unintentionally, to
    plants. The conversion products that have been identified (Annex
    Fig. 6) are DDE, TDE (DDD), DDMU, DDA, conjugates of DDA, and a
    conjugate of DBH (Zimmer & Klein, 1972), which means that the
    metabolites in plants are not chemically different from those in other
    organisms.

        In a study of the accumulation and distribution of  p,p'-DDT in
    an apple orchard, DDT residues in or on the roots and leaves of the
    herbage and the roots, bark, leaves, and fruit of the trees were
    recorded for an orchard sprayed annually (Stringer et al., 1975).
    During 13 years, there were increasing amounts of DDE, TDE, and DDMU
    in relation to DDT, in the bark of apple trees indicating some
    breakdown on the bark (< 10%). DDE and TDE were also observed after
    application of  p,p'-DDT to cotton (Nash et al., 1977). These 2
    substances seem to be common conversion products of DDT in plants. The
     o,p'-DDT observed in the last 2 experiments seems to be an impurity
    of the DDT rather than a metabolite.

    2.4  Microorganisms and soil

        The most common metabolic reaction of DDT in microorganisms is
    reductive dechlorination resulting in the formation of TDE. This
    reaction has been demonstrated in  Escherichia coli (in rat
    intestine),  Aerobacter aerogenes, Proteus vulgaris, and in yeasts.
    In contrast to metabolism in higher animals, dechlorination by
    microorganisms is anaerobic and is catalysed by reduced cytochrome
    oxidase (EC 1.9.3.1). Fe (II)-cytochrome oxidase isolated from
     Aerobacter converts DDT to TDE  in vitro (Klein & Korte, 1970). The
    conversion of DDT to TDE (DDD) in bodies of water (Miskus et al.,
    1965) and in other reducing environments characteristic of dead and

    

    decaying matter (Zoro et al., 1974) is mediated by reduced iron
    porphyrins and is not an essential part of cell metabolism. These
    findings have considerable environmental significance since most
    living material contains iron porphyrins bound with protein in complex
    molecules. The porphyrins are released after decay of the organic
    substances, and may then be regarded as widespread environmental
    agents that convert, on a larger scale, the persistent DDT to the less
    persistent TDE. TDE is susceptible to further abiotic or biotic
    degradation.

        However, the formation of DDE and DDA from DDT by microorganisms
    is also possible. For instance, both DDE and TDE were isolated from
     Serratia marcescens and Alkaligenes faecalis (Stenersen, 1965) and
    DDA was isolated from microbial cultures obtained from agricultural
    soil (Patil et al., 1970).

        In a model experiment with anaerobic activated sludge and  p,p'-
    DDT-14C, TDE,  p,p'-dichlorobenzophenone, DDMU and a so far unknown
    metabolite, DDCN (bis[ p-chlorophenyl] acetonitrile), were detected
    as conversion products. The last of these substances, a minor
    conversion product, was also found in the sediment layer of the Lake
    Mälaren in Sweden (0.2 mg/kg dry weight). DDCN is formed via TDE or
    DDE, but directly from DDT (Jensen et al., 1972).

        The question of "bound residues" in soil, which is now under
    discussion for a number of "non-persistent" pesticides, especially
    those that are anilin-derived, seems also to be relevant for
    "persistent" substances such as DDT, although the percentage of bound
    residues is less than for less persistent pesticides. The formation of
    25% of bound DDT-residues within 28 days (Lichtenstein et al., 1977)
    justifies a reassessment of the persistence of DDT in soil. Further
    information should be obtained concerning the nature and the potential
    biological activity of the compounds that are bound.

    3  Conclusion

        A multitude of conversion products are formed from DDT under
    environmental conditions. Nearly 20 of these (including mammalian
    metabolites) have been identified so far, but the chemical structure
    of a number of other compounds is still unknown. Very little is known
    of the toxicological properties of these conversion products with the
    exception of major products such as DDE and TDE. This should be
    remembered when the unwanted effects of DDT in the environment are
    evaluated. However, there is even less information concerning the fate
    in the environment of many other pesticides including those that are
    used as DDT-substitutes.

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