Polychlorinated dibenzo-p-dioxins and dibenzofurans (EHC 88, 1989)

INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

ENVIRONMENTAL HEALTH CRITERIA 88

POLYCHLORINATED DIBENSO- PARA-DIOXINS AND DIBENZOFURANS

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CONTENTS

ENVIRONMENTAL HEALTH CRITERIA FOR POLYCHLORINATED
DIBENZO-PARA-DIOXINS AND DIBENZOFURANS

1. SUMMARY AND RECOMMENDATIONS

1.1. Summary
1.1.1. Sources
1.1.2. Ambient levels and routes of exposure
1.1.3. Toxicokinetics, biotransformation, and
biological monitoring
1.1.4. Health effects
1.1.4.1 Animals
1.1.4.2 Humans
1.1.5. Conclusion
1.2. Recommendations

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES,
ANALYTICAL METHODS
2.1. Identity
2.2. Physical and chemical properties
2.3. Analytical methods
2.3.1. General aspects
2.3.2. Sampling strategy and sampling methods
2.3.3. Extraction procedures
2.3.4. Sample clean-up
2.3.5. Isomer identification
2.3.6. Quantification
2.3.7. Confirmation
2.3.8. Other analytical methods

3. SOURCES OF ENVIRONMENTAL POLLUTION
3.1. Production, synthesis, and use
3.2. Industrial processes
3.3. Contamination of commercial products
3.3.1. Chlorophenoxyacetic acid herbicides
3.3.2. Hexachlorophene
3.3.3. Chlorophenols
3.3.4. Polychlorinated biphenyls (PCBs)
3.3.5. Chlorodiphenyl ether herbicides
3.3.6. Hexachlorobenzene
3.3.7. Rice oil
3.4. Sources of heavy environmental pollution
3.4.1. Industrial accidents
3.4.2. Improper disposal of industrial waste
3.4.3. Heavy use of chemicals
3.5. Other sources of PCDDs and PCDFs in the
environment
3.5.1. Thermal degradation of technical
products
3.5.2. Incineration of municipal waste

3.5.3. Incineration of sewage sludge
3.5.4. Incineration of hospital waste
3.5.5. Incineration of hazardous waste
3.5.6. Metal industry and metal treatment
industry
3.5.7. Wire reclamation
3.5.8. Traffic
3.5.9. Fires and accidents in PCB-filled
electrical equipment
3.5.10. Pulp and paper industry
3.5.11. Incineration of coal, peat, and wood
3.5.12. Inorganic chlorine precursors
3.5.13. Photochemical processes
3.6. Comparison of isomeric pattern and congener
profiles from various sources

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND
TRANSFORMATIONS

4.1. Environmental transport
4.1.1. Air
4.1.2. Water
4.1.3. Soil and sediments
4.2. Environmental transformation
4.2.1. Abiotic transformation
4.2.2. Biotransformation and biodegradation
4.3. Bioaccumulation
4.4. Levels in biota
4.4.1. Vegetation
4.4.2. Aquatic organisms
4.4.3. Terrestrial animals
4.4.4. Human data
4.4.4.1 Adipose tissue
4.4.4.2 Blood plasma

5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

5.1. Air
5.2. Water and leachate
5.3. Soil and sediment
5.4. Food
5.4.1. Meat and bovine milk
5.4.2. Human milk
5.4.3. Rice
5.5. Yusho and Yu-cheng episodes

6. KINETICS AND METABOLISM OF 2,3,7,8-TETRACHLORODIBENZO-
P-DIOXIN (TCDD) AND OTHER PCDDs

6.1. Uptake, distribution, and excretion
6.1.1. Studies on rats
6.1.2. Studies on mice

6.1.3. Studies on guinea-pigs
6.1.4. Studies on hamsters
6.1.5. Studies on monkeys
6.1.6. Studies on dogs
6.1.7. Studies on cows
6.1.8. In vitro studies
6.2. Metabolic transformation
6.2.1. Studies on mammals
6.2.1.1 In vivo studies
6.2.1.2 In vitro studies
6.3. Transfer via placenta and/or milk
6.4. Matrix effects on the uptake
("bio-availability")

7. EFFECTS OF TCDD AND OTHER PCDDs ON EXPERIMENTAL
ANIMALS AND IN VITRO TEST SYSTEMS

7.1. Acute toxicity
7.1.1. In vivo studies on mammals
7.1.2. In vitro studies on mammalian cells
7.1.3. Studies on birds
7.1.4. Toxicity of metabolites
7.1.5. Modulation of the acute toxicity
7.2. Short-term toxicity
7.2.1. Studies on rats
7.2.2. Studies on mice
7.2.3. Studies on guinea-pigs
7.2.4. Studies on hamsters
7.2.5. Studies on monkeys
7.3. Long-term toxicity
7.3.1. Studies on rats
7.3.2. Studies on mice
7.3.3. Studies on monkeys
7.4. Effects detected by special studies
7.4.1. Wasting syndrome
7.4.2. Hepatotoxicity
7.4.2.1 Morphological alterations
7.4.2.2 Hepatic plasma membrane
function
7.4.2.3 Biliary excretion
7.4.3. Porphyria
7.4.4. Epidermal effects
7.4.4.1 In vivo studies
7.4.4.2 In vitro studies
7.4.5. Effects on the immune system
7.4.5.1 Histopathology
7.4.5.2 Humoral-mediated immunity
7.4.5.3 Cell-mediated immunity
7.4.5.4 Macrophage function
7.4.6. Myelotoxicity
7.4.7. Effects on the intermediary
metabolism

7.4.8. Enzyme induction
7.4.8.1 Studies on rats
7.4.8.2 Studies on mice
7.4.8.3 Studies on guinea-pigs
7.4.8.4 Studies on rabbits
7.4.8.5 Studies on hamsters
7.4.8.6 Studies on cows
7.4.8.7 Studies on chick embryos
7.4.8.8 Studies on cell cultures
7.4.9. Endocrine effects
7.4.10. Vitamin A storage
7.5. Embryotoxicity and reproductive effects
7.5.1. Studies on rats
7.5.2. Studies on mice
7.5.3. Studies on rabbits
7.5.4. Studies on monkeys
7.5.5. Studies on chickens
7.6. Mutagenicity and related end-points
7.6.1. Mutagenicity
7.6.1.1 Studies on bacteria
7.6.1.2 Studies on eukaryotic cells
7.6.1.3 In vivo studies
7.6.2. Interaction with nucleic acids
7.6.3. Cytogenetic effects
7.6.4. Cell transformation
7.7. Carcinogenicity
7.7.1. Long-term animal studies on single
compounds
7.7.2. Long-term animal studies with mixed
compounds
7.7.3. Short-term and interaction studies
7.8. Mechanisms of action
7.8.1. Receptor-mediated effects
7.8.2. Toxicokinetics
7.8.3. Impairment of normal cellular regulatory
systems
7.8.3.1 Endocrine imbalance
7.8.3.2 Body weight regulation
7.8.3.3 Plasma membrane function
7.8.3.4 Impaired vitamin A storage
7.8.4. Lipid peroxidation

8. EFFECTS OF PCDDs ON HUMAN BEINGS - EPIDEMIOLOGICAL
AND CASE STUDIES

8.1. Occupational studies - historical perspective
8.2. General population studies
8.2.1. Missouri, USA
8.2.2. Seveso, Italy
8.2.3. Viet Nam
8.3. Signs and symptoms in humans associated with
TCDD exposure

8.3.1. Skin manifestations
8.3.2. Systemic effects
8.3.3. Neurological effects
8.3.4. Psychiatric effects
8.4. Epidemiological studies
8.5. Human experimental studies

9. TOXICOKINETICS OF PCDFs

9.1. Uptake, distribution, and excretion
9.1.1. Studies with 2,3,7,8-tetrachlorodibenzo-
furan (2,3,7,8-TCDF)
9.1.2. Studies with other PCDFs
9.2. Metabolic transformation
9.3. Transfer via placenta and/or milk

10. EFFECTS OF PCDFs ON ANIMALS

10.1. Acute toxicity
10.1.1. Studies on rats
10.1.2. Studies on mice
10.1.3. Studies on guinea-pigs
10.1.4. Studies on rabbits
10.1.5. Studies on monkeys
10.2. Short-term toxicity
10.2.1. Studies on rats
10.2.2. Studies on mice
10.2.3. Studies on guinea-pigs
10.2.4. Studies on rabbits
10.2.5. Studies on hamsters
10.2.6. Studies on monkeys
10.2.7. Studies on chickens
10.3. Chronic toxicity
10.3.1. Studies on monkeys
10.4. Effects detected by special studies
10.4.1. Immunobiological effects
10.4.1.1 Histopathology
10.4.1.2 Humoral-mediated immunity
10.4.1.3 Cell-mediated immunity
10.4.2. Enzyme induction
10.4.2.1 Studies on rats
10.4.2.2 Studies on mice
10.4.2.3 Studies on chickens
10.4.2.4 Studies on cell cultures
10.4.3. Receptor binding
10.5. Embryotoxicity and reproductive effects
10.6. Mutagenicity
10.7. Carcinogenicity

11. EFFECTS OF PCDFs ON HUMAN BEINGS

11.1. Yusho and Yu-cheng

12. EVALUATION OF HEALTH RISKS FROM THE EXPOSURE TO
CHLORINATED DIBENZO-P-DIOXINS (PCDDs) AND
DIBENZOFURANS (PCDFs)

12.1. Introduction
12.2. Exposure assessment
12.2.1. Sources of contamination
12.2.2. Ambient levels
12.2.3. Routes of exposure
12.2.4. Bioavailability
12.3. Animal data
12.3.1. Toxicokinetics of 2,3,7,8-TCDD
12.3.2. Toxicokinetics of PCDDs and PCDFs,
other than TCDD
12.3.3. Toxic effects 2,3,7,8-TCDD
12.3.4. Toxic effects of PCDDs and PCDFs,
other than TCDD
12.3.5. Review of species differences
12.4. Human health effects
12.4.1. PCDDs
12.4.2. PCDFs
12.4.3. Human body burden and kinetics
12.5. General conclusions

13. RECOMMENDATIONS

14. EVALUATIONS BY INTERNATIONAL BODIES AND THE CONCEPT
OF TCDD EQUIVALENTS

14.1. International evaluations
14.2. Methodologies used in assessment of
risk from PCDDs and PCDFs
14.2.1. Individual congeners
14.2.2. Mixtures of PCDD and PCDF congeners and
isomers - concept of TCDD toxic
equivalents

REFERENCES

FRENCH TRANSLATION OF SUMMARY, EVALUATION, AND
RECOMMENDATIONS

WHO TASK GROUP ON CHLORINATED DIBENZO-p-DIOXINS AND
DIBENZOFURANS

Members

Dr U.G. Ahlborg, Unit of Toxicology, National Institute of
Environmental Medicine, Stockholm, Sweden
Dr J.S. Bellin, Office of Toxic Substances, US Environmental
Protection Agency, Washington, DC, USA
Dr B. Birmingham, Ministry of the Environment, Hazardous Contaminants
Section, Toronto, Ontario, Canada
Professor A.D. Dayan, Department of Health and Social Security,
St Bartholomew's Hospital Medical College, London, United
Kingdom (Chairman)
Dr A. di Domenica, Instituto Superiore di Sanita, Rome, Italy
Dr M. Greenberg, Department of Health and Social Security,
Division of Toxicology and Environmental Protection, London,
United Kingdom
Dr R.D. Kimbrough, United States Department of Health and Human
Services, Center for Disease Control, Atlanta, Georgia, USA
(Now at the US Environmental Protection Agency Washington,
DC, USA)
Dr R. Koch, Department of Toxicology, Institute of Hygiene,
Gera, DDR
Professor C. Rappe, Department of Chemistry, University of
Umea, Umea, Sweden
Dr S. Safe, Texas A and M University, College Station, Texas,
USA
Dr H. Spielmann, Max von Pettenkofer Institute, Bundesgesundheitsamt,
Berlin (West)
Dr J. Vos, National Institute of Public Health and Environmental
Hygiene, Bilthoven, Netherlands

Representatives

Dr A. Berlin, Health and Safety Directorate, Commission of the
European Communities, Luxembourg
Mrs E. Cox, Department of the Environment, London, United
Kingdom
Miss F.D. Pollitt, Department of the Environment, London,
United Kingdom

Secretariat

Dr G.C. Becking, International Programme on Chemical Safety,
World Health Organization, Research Triangle Park, North
Carolina, USA (Secretary)

Secretariat (contd)

Dr H. Hakensson, Unit of Toxicology, National Institute of
Environmental Medicine, Stockholm, Sweden (Temporary
Adviser) (Rapporteur)
Dr E. Johnson, International Agency for Research on Cancer,
World Health Organization, Lyons, France
Dr S. Tarkowski, Regional Office for Europe, World Health
Organization, Copenhagen, Denmark

NOTE TO READERS OF THE CRITERIA DOCUMENTS

Every effort has been made to present information in the criteria
documents as accurately as possible without unduly delaying their
publication. In the interest of all users of the environmental health
criteria documents, readers are kindly requested to communicate any
errors that may have occurred to the Manager of the International
Programme on Chemical Safety, World Health Organization, Geneva,
Switzerland, in order that they may be included in corrigenda, which
will appear in subsequent volumes.

* * *

A detailed data profile and a legal file can be obtained from the
International Register of Potentially Toxic Chemicals, Palais des
Nations, 1211 Geneva 10, Switzerland (Telephone No. 7988400 -
7985850).

ENVIRONMENTAL HEALTH CRITERIA FOR POLYCHLORINATED DIBENZO-PARA-
DIOXINS AND DIBENZOFURANS

A WHO Task Group on Environmental Health Criteria for
Polychlorinated Dibenzo-para-dioxins and Dibenzofurans met at the
Monitoring and Assessment Research Centre, London, United Kingdom,
from 9 to 13 February, 1987. Dr M. Berlin opened the meeting and
welcomed the members on behalf of the host Institute and on behalf of
the United Kingdom Department of Health and Social Security, who
sponsored the meeting. Dr G.C. Becking addressed the meeting on behalf
of the three cooperating organizations of the IPCS (UNEP, ILO, and
WHO). The Task Group reviewed and revised the draft criteria document
and made an evaluation of the risks for human health and for the
environment from exposure to polychlorinated dibenzo-p-dioxins and
dibenzofurans.

The drafts of this document were prepared by Dr U.G. Ahlborg, Dr
H. Hakensson, and Dr B. Holmstedt, all of the National Institute of
Environmental Medicine, Stockholm, Sweden, and by Professor C. Rappe
of the University of Umea, Umea, Sweden.

The efforts of all who helped in the preparation and finalization
of the document are gratefully acknowledged.

* * *

Partial financial support for the publication of this criteria
document was kindly provided by the United States Department of Health
and Human Services, through a contract from the National Institute of
Environmental Health Sciences, Research Triangle Park, North Carolina,
USA - a WHO Collaborating Centre for Environmental Health Effects. The
United Kingdom Department of Health and Social Security generously
supported the cost of printing.

ABBREVIATIONS

AHH aryl hydrocarbon hydroxylase
ALA aminolevulinic acid
BGG bovine gammaglobulin
BHA butylated hydroxyanisole
BP benzo(a)-pyrene
CMI cell-mediated immunity
DEN diethylnitrosamine
diCDD dichlorinated dibenzo-p-dioxin
diCDF dichlorinated dibenzofuran
DMBA dimethylbenzathraline
ECOD 7-ethoxycoumarin-o-deethylase
EGF epidermal growth factor
EH epoxide hydratase
EI electron impact
EROD 7-ethoxyresurofin-o-deethylase
ETG epidermal transglutaminase
fg femtogram (10^-15g)
GC gas chromatography
heptaCDD heptachlorinated dibenzo-p-dioxin
heptaCDF heptachlorinated dibenzofuran
hexaCDD hexachlorinated dibenzo-p-dioxin
hexaCDF hexachlorinated dibenzofuran
HMI humoral-mediated immunity
HPLC high pressure liquid chromatography
IARC International Agency for Research on Cancer
ip intraperitoneal
IR infrared
LOEL lowest-observed-effect level
MCPA 4-chloro-o-tolyloxyacetic acid
MFO mixed-function oxidase
MS mass spectrometry
MSW municipal solid waste
ng nanogram (10^-9g)
NMR nuclear magnetic resonance
NOEL no-observed-effect level
octaCDD octachlorinated dibenzo-p-dioxin
octaCDF octachlorinated dibenzofuran
PAH polyaromatic hydrocarbons
PCB polychlorinated biphenyl
PCDD polychlorinated dibenzo-p-dioxin
PCDF polychlorinated dibenzofuran
PCDPE polychlorinated diphenylether
PCPY polychlorinated pyrene
PCQ polychlorinated quaterphenyl
pentaCDD pentachlorinated dibenzo-p-dioxin
pentaCDF pentachlorinated dibenzofuran
pg picogram (10-¹²g)
SC subcutaneous
SCE sister chromatid exchange

SD standard deviation
SEM standard error of the mean
SIM selected ion monitoring
TCDD 2,3,7,8-tetrachlorinated dibenzo-p-dioxin
TCDF 2,3,7,8-tetrachlorinated dibenzofuran
TCP trichlorophenol
tetraCDD tetrachlorinated dibenzofuran
tetraCDF tetrachlorinated dibenzofuran
TPA 12-o-tetradecanoylphorbol-13-acetate
triCDD trichlorinated dibenzo-p-dioxin
triCDF trichlorinated dibenzofuran
t3 triiodothyronine
t4 thyroxine
UDPGT UDP-glucuronosyltransferase
UV ultraviolet
2,4-D 2,4-dichlorophenoxyacetic acid
2,4,5-T 2,4,5-trichlorophenoxyacetic acid
3-MC 3-methylcholanthrene

1. SUMMARY AND RECOMMENDATIONS

1.1 Summary

1.1.1 Sources

Polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated
dibenzofurans (PCDFs) are two series of tricyclic aromatic compounds
with similar chemical and physical properties; they are ubiquitous in
the environment. They do not occur naturally, nor are they
intentionally produced. There are 75 positional isomers of PCDDs and
135 isomers of PCDFs.

The most important sources of contamination with PCDDs and PCDFs
include:
- contaminated commercial chemical products, such as
chlorinated phenols and their derivatives, and PCBs;
- incineration of municipal, hazardous, and hospital
wastes, and of sewage sludges;
- automobile operation;
- fossil fuel combustion;
- overheating and emissions from fires involving PCBs;
- disposal of industrial wastes resulting from
processes such as the production of chlorophenols and
their derivatives, chlorophenol wood treatment, use
of PCB fluids in electrical equipment, and wastes
from pulp and paper processing.

1.1.2 Ambient levels and routes of exposure

The limited data available indicate that ambient levels of these
compounds are very low in air, soil, and sediment, i.e. fg/m³ in
air, ng/kg in soil and sediment. Levels of PCDDs and PCDFs up to 50
ng/kg have been found in aquatic organisms in the general environment.
Data on contamination of drinking water and commercial food are very
limited.

Exposure to these compounds in the general population probably
occurs mainly through the food-chain.

Some workers engaged in the production, use, and destruction of
materials containing PCDDs and PCDFs and their precursors may receive
high exposure. For these persons, inhalation and dermal contact are
the primary exposure routes of concern.

1.1.3 Toxicokinetics, biotransformation, and biological monitoring

The bioavailability of PCDDs and PCDFs depends on the matrix they
are in and the route of exposure. Data on bioavailability through
inhalation are not available for any species.

The quantity absorbed by humans after any route of exposure is
not known.

Studies on rodents given single or repeated oral doses of
2,3,7,8-TCDD have shown that about half of the administered dose is
absorbed from the gastrointestinal tract. The reported half-lives for
elimination were between 12 and 94 days for rodents. The half-life of
2,3,7,8-TCDD in adipose tissue of the rhesus monkey is about 1 year.

Animal data on the toxicokinetics of PCDDs other than
2,3,7,8-TCDD are limited. The half-life for 2,3,7,8-TCDD has been
reported to be in the range of 2 and 8 days for rats, mice, and
monkeys and more than 20 days for guinea-pigs. Studies on rats have
shown that 2,3,4,7,8-pentaCDF is more highly retained than is
2,3,7,8-TCDD.

Data on the retention of PCDDs and PCDFs in tissues of various
species, exposed to synthetic mixtures or to environmental samples
containing PCDDs and PCDFs, show a high variability in retention time
between congeners with or without chlorine substitution in the 2,3,7,
and 8 positions.

Limited human data indicate half-lives for some 2,3,7,8-
substituted PCDDs and PCDFs in the range of 2-6 years.

The PCDDs and PCDFs are predominately stored in fat, but they are
also excreted in milk and pass through the placenta. They also appear
in the blood and vital organs at lower concentrations.

The tissue distribution in humans is not clear at present,
although it has been suggested that the ratio between fatty tissue and
liver is higher in humans than in rodents.

In human fat, background levels of TCDD up to 20 ng/kg have been
found in the general population, with no known specific exposure, but
higher levels have been reported in some cases without evidence of
disease. None of these populations were randomly sampled. The more
highly chlorinated PCDDs and PCDFs, particularly octaCDD, are also
present in these samples. Average tissue levels of TCCD tend to
increase with age.

1.1.4 Health effects

1.1.4.1 Animals

The toxic and biological effects resulting from exposure to
2,3,7,8-TCDD are dependent on a number of factors, which include the
species, strain, age, and sex of the animals used. The toxic responses
observed in several animal species include body weight loss,
hepatotoxicity, porphyria, dermal toxicity, gastric lesions, thymus

atrophy and immunotoxicity, teratogenicity, reproductive effects, and
carcinogenicity. TCDD induces a wide spectrum of biological effects
including enzyme induction and vitamin A depletion. Not all of these
effects are observed in any single animal species. The most
characteristic toxic effects observed in all laboratory animals are
body weight loss, thymus atrophy, and immunotoxicity. Chloracne and
related dermal lesions are the most frequently noted signs of
2,3,7,8-TCDD toxicosis in humans; dermal lesions are also observed in
rhesus monkeys, hairless mice, and rabbits. In contrast, most rodents
do not develop chloracne and related dermal toxic lesions after
exposure to 2,3,7,8-TCDD. Many of the toxic lesions are noted
primarily in epithelial tissues.

Reproductive effects have been reported in rhesus monkeys and
rats. The lowest-observed-effect levels have been reported to be
approximately 1-2 ng/kg body weight per day. In two cancer studies in
rats, hepatocellular carcinomas were produced at approximate dose
levels of 0.1 µg/kg body weight per day and 0.01 µg/kg body weight per
day. Doses of 0.001 µg/kg body weight resulted in foci or areas of
hepatocellular alteration. The incidence of certain hormone-dependent
tumours was lower than in the control animals.

TCDD does not appear to have mutagenic properties, and is
therefore not likely to be genotoxic. Thus, it is assumed to be
carcinogenic through an indirect mechanism.

Several other PCDDs and PCDFs cause signs and symptoms similar to
those of 2,3,7,8-TCDD, but there is a wide variation with regard to
potency. There are 12 isomers that display higher toxicity, i.e., the
tetra-, penta-, hexa-, and heptaCDDs and CDFs with four chlorine atoms
in the symmetrical lateral positions 2,3,7, and 8. A mixture of two
hexachlorodibenzo-p-dioxins (1,2,3,7,8,9- and 1,2,3,6,7,8-hexaCDD)
has been demonstrated to possess carcinogenic properties in long-term
animal studies, but at higher doses than those used in the study of
TCDD. Dibenzo-p-dioxin and 2,7-diCDD failed to demonstrate
carcinogenic properties. The relative toxic and biological potencies
of PCDDs and PCDFs have been estimated using short-term studies in
rats and mammalian cell cultures.

There are marked species differences in the susceptibility of
animals to the biological and toxic effects elicited by
2,3,7,8-substituted PCDDs and PCDFs. For example, the oral LD50 values
range from 0.6 µg/kg body weight in guinea-pigs, to 5051 µg/kg body
weight in Golden Syrian hamsters for 2,3,7,8-TCDD. The tremendous
variation in species and strain sensitivity to 2,3,7,8-TCDD and
related compounds cannot be explained by the observed toxicokinetic
differences. The toxicity and toxicokinetics of TCDD in monkeys most
closely resemble the effects observed in humans. There is evidence in

inbred mice that the cellular levels of the Ah receptor correlate, in
part, with susceptibility to the biological and toxic effects of these
compounds. The receptor has also been identified in other species
including man. However, interspecies comparison of cellular Ah
receptor levels do not explain fully the differences in sensitivity.

1.1.4.2 Humans

For occupational and accidental exposures to PCDDs and PCDFs, in
spite of many clinical and follow-up studies, no clear-cut persistent
systemic effects have been delineated except for chloracne. Other
effects have been noted, but, apart from chloracne and perhaps minor
functional disorders, none has been persistent.

In some epidemiological studies of people exposed to a mixture of
dioxins, furans, and other chemicals, an increased incidence of cancer
at different sites has been claimed, but a number of factors limits
confidence in the findings.

In the Seveso accident, the only clear-cut adverse health effect
recorded has been chloracne. Chloracne (193 cases) occurred in 1976
and 1977, and 20 of those individuals still had active chloracne in
1984. Many studies have been performed to find possible links between
exposure to Agent Orange and health effects in civilians or military
personnel in Viet Nam. However, the information available to date does
not allow definite conclusions to be drawn with regard to effects on
human reproduction or any other significant health effects.

In the Missouri incident, children who showed acute illness when
the contamination occurred in 1971 are now reportedly in good health.
Furthermore, epidemiological studies in Missouri on populations
exposed to lower concentrations of dioxins over longer periods of time
have so far not revealed any significant health effects. Although no
clinical symptoms were observed, there were indications of an effect
on the cell-mediated immune system.

The only documented intoxications with PCDFs in humans are the
two instances of contamination of rice oil with PCDFs, PCBs, and PCQs,
i.e., Yusho in Japan, 1968, and Yu-cheng in Taiwan, 1979. In total,
several thousand people were acutely intoxicated. From the data it
appears most likely that the causative agent was the PCDFs. The
general symptomatology was similar to that seen in intoxications with
TCDD, with the differences reflecting the intensity of exposure and
the ages and sex of those exposed.

The average daily intake of 2,3,7,8-substituted PCDFs by Yusho
patients was estimated to be 0.1-0.2 µg/kg body weight for a period of
several months, while the lowest dose causing disease was estimated to
be 0.05-0.1 µg/kg body weight per day over a period of 30 days.

1.1.5 Conclusion

PCDDs and PCDFs occur throughout the environment and we all
probably carry a body burden of them. They have sometimes produced
complex toxic effects following occupational and accidental exposure.

Based on the Yusho disease and experiments in sensitive species
of monkeys, and making assumptions about the relative potencies of
PCDDs and PCDFs, man and certain monkeys may have comparable
sensitivity to these compounds. However, the uncertainties related to
the real dose received by humans and the difficulties of assessing
toxic effects other than chloracne in humans prevents a firm
conclusion as to the relative resistance of humans to the toxic
effects of these compounds. Exposure should be reduced to levels as
low as reasonably practicable.

1.2 Recommendations

1. Analytical interlaboratory validation and "round-robin" studies
using standardized quality assurance and quality control procedures
are needed to improve analytical methodology.

Sampling strategy and analytical procedures and data
interpretation should be optimized and standardized before undertaking
surveys.

2. Further information is required about the origins and
environmental distributon and fate of PCDDs and PCDFs.

Further monitoring data, including time trends and determinations
of isomer patterns, are required for environmental levels of PCDDs and
PCDFs, especially for food, ambient air, and sediments.

3. Data should be obtained about the effects of PCDDs and PCDFs on
environmental biota.

4. More information is required on the bioavailability of PCDDs and
PCDFs from different matrices in the environment and from the diet.
Exposure from these sources should be correlated with agricultural and
industrial practices.

5. Simpler and less expensive chemical and biological methods
suitable for screening for the presence of PCDDs and PCDFs should be
developed and validated.

6. Studies to determine the mechanisms of toxicity of PCDDs and
PCDFs are needed to support an evaluation of the differences in
effects between species and to support an extrapolation to man.

7. Further investigation of immunotoxicity is important, including
cytotoxic T-lymphocyte function. Studies of the effects of perinatal
exposure and of the duration of actions on the immune system are
important.

8. Long-term toxicity studies should be carried out, including
multigeneration reproductive studies in different species with three
of the most widespread PCDDs and PCDFs, namely 2,3,4,7,8-pentaCDF,
1,2,3,7,8-pentaCDD, and octaCDD.

9. Because humans are exposed to complex mixtures of PCDDs and
PCDFs, test systems, including human cell culture systems, should be
developed further and validated for evaluating the toxic potency of
these compounds and other mixtures. These systems can be used to study
mechanisms of action, structure activity relationships, and
interactive effects.

10. Investigations to examine the body burden and to correlate it
with clinical effects and laboratory findings are indicated. Follow-up
studies of previously exposed groups are important.

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS

2.1 Identity

The polychlorinated dibenzo-para-dioxins (PCDDs) and
polychlorinated dibenzofurans (PCDFs) are two series of almost planar
tricyclic aromatic compounds with very similar chemical properties.
The general formulae are given in Fig. 1.

The number of chlorine atoms can vary between 1 and 8. The term
isomers refers to comparisons between compounds with the same
empirical formulae. The term congeners refers to comparisons between
compounds within the same series but with a different number of
chlorine atoms. The number of positional isomers is quite large; in
all there are 75 PCDDs and 135 PCDFs and the number of isomers for a
certain number of chlorine atoms is given in Table 1.

The nomenclature used in this document is based on the system
used by Chemical Abstracts. The Chemical Abstracts System Registry
Numbers (CAS RN) for a few PCDDs and PCDFs that have been cited in the
literature are provided in Table 2.

2.2 Physical and Chemical Properties

A large number of the individual PCDDs have been synthesized by
various methods and characterized, mainly by gas chromatography-mass
spectrometry (GC/MS) (Buser & Rappe, 1980, 1984; Taylor et al., 1985;
Rappe et al., 1985a) but also by using nuclear magnetic resonance
(NMR) or ultraviolet (UV), infrared (IR), (Pohland & Yang, 1972; Kende
et al., 1974), or X-ray analyses (Boer et al., 1973; Slonecker et al.,
1983).

Table 1. Number of PCDD and PCDF isomers

Number Number Number
of chlorine atoms of PCDD isomers of PCDF isomers

1 2 4
2 10 16
3 14 28
4 22 38
5 14 28
6 10 16
7 2 4
8 1 1
75 135

Table 2. CAS RN for some PCDDs and PCDFs

PCDD congener CAS RN PCDF congener CAS RN

2,3,7,8-TetraCDD 1746-01-6 2,3,7,8-TetraCDF 51207-31-9
1,2,3,7,8-PentaCDD 40321-76-4 1,2,3,7,8-PentaCDF 57117-41-6
1,2,3,6,7,8-HexaCDD 57653-85-7 2,3,4,7,8-PentaCDF 57117-31-4
1,2,3,7,8,9-HexaCDD 19408-74-3 1,2,3,4,7,8-HexaCDF 70648-26-9
1,2,3,4,6,7,8-HeptaCDD 35822-46-9 1,2,3,6,7,8-HexaCDF 57117-44-9
1,2,3,4,7,8,9-HeptaCDD 58200-70-7 1,2,3,7,8,9-HexaCDF 72918-38-8
OctaCDD 3268-87-9 2,3,4,6,7,8-HexaCDF 60851-34-5

Pyrolysis of chlorinated phenols yields small amounts of one or
more PCDD isomers. Using this technique all the 22 tetraCDDs have been
prepared (Nestrick et al., 1979; Buser & Rappe, 1980) as well as the
14 pentaCDDs (Buser & Rappe, 1984) and 10 hexaCDDs (Lamparski &
Nestrick, 1981; Buser & Rappe, 1984).

Taylor et al. (1985) have synthesized, separated, and isolated
all the 22 tetraCDD isomers. In Table 3 are listed some other isomers
that have been synthesized and isolated.

The most toxic and most extensively studied representative of the
chlorinated dioxins (PCDDs) is
2,3,7,8-tetrachlorodibenzo-para-dioxin (2,3,7,8-tetraCDD) (Fig. 2).
It is commercially available, as are more than 10 other PCDD
congeners.

The empirical formulae, molecular weights, and some physical
properties of a few PCDDs are given in Table 4.

Table 3. Synthetic method and melting point for some PCDDs

PCDD Synthetic Melting point Reference
Isomer method^a °C

1-Chloro- 1 80-90 Pohland & Yang, 1972
2-Chloro- 1 88-89 Pohland & Yang, 1972
1,3-Dichloro- 1 113.5-114.5 Kende et al., 1974
2,3-Dichloro- 1 163-164 Pohland & Yang, 1972
2,7-Dichloro- 2 209-210 Pohland & Yang, 1972
2,8-Dichloro- 3 150.5-151 Pohland & Yang, 1972
1,2,4-Trichloro- 4 128-129 Pohland & Yang, 1972
2,3,7-Trichloro- 1 157-158 Kende et al., 1974
2,3,7,8-Tetrachloro- 2 305-306 Pohland & Yang, 1972
2,3,7,8-Tetrachloro- 5 305-307 Kende et al., 1974
1,2,3,4-Tetrachloro- 4 188-190 Pohland & Yang, 1972
1,3,7,8-Tetrachloro- 1 193.5-195 Kende et al., 1974
1,3,6,8-Tetrachloro- 2 219-219.5 Pohland & Yang, 1972
1,2,3,4,7-Pentachloro- 5 195-196 Kende et al., 1974
1,2,3,4,7,8-Hexachloro- 5 275 Pohland & Yang, 1972
1,2,4,6,7,9-Hexachloro- 2 238-240 Pohland & Yang, 1972
Octachloro 2 330 Pohland & Yang, 1972

^a Synthetic methods as follows:

1 = Catechol + chlorobenzene
2 = Pyrolysis of chlorphenols
3 = Cyclization of chlorophenoxyphenol
4 = Catechol + chloronitrobenzene
5 = Chlorination of chlorodibenzodioxin

Table 4. Physical properties of some PCDDs

Molecular Molecular Absorption
Compound formulae weight maximum Reference
(chloroform)
(nm)

2,3,7,8-TCDD C₁₂H₄Cl₄O₂ 321.9 310 Pohland &
Yang (1972)

1,2,3,7,8-PentaCDD C₁₂H₃Cl₅O₂ 356.5 308 Gray et al.
(1976)

1,2,3,6,7,8-HexaCDD C₁₂H₂Cl₆O₂ 390.9 316 Gray et al.
(1975)

1,2,3,7,8,9-HexaCDD C₁₂H₂Cl₆O₂ 390.9 317 Gray et al.
(1975)

Although tetraCDD is lipophilic, it is only slightly soluble in
most solvents and very slightly soluble in water (Table 5).

Table 5. Solubility of 2,3,7,8-tetraCDD in various solvents^a

Solvent Solubility at 25 °C
g/litre g/kg

O-Dichlorobenzene 1.8 1.4
Chlorobenzene 0.8 0.72
Perchloroethylene 0.68 0.48
Chloroform 0.55 0.37
Benzene 0.47 0.57
Acetone 0.09 0.11
Dimethylsulfoxide^b < 0.1 < 0.1
Methanol 0.01 0.01
Water 2 x 10^-7 2 x 10^-7

^a From: Crummett & Stehl (1973).
^b DMSO caused detector fouling and a better value could not be obtained.

Table 6. Water solubility of PCDDs^a

Compound Water solubility (g/litre)
20.0 °C 40.0 °C

1,3,6,8-TetraCDD (3.2±0.2) x 10^-7 (3.9±0.4) x 10^-7
1,2,3,7-TetraCDD (4.3±0.1) x 10^-7 (12.7±0.8) x 10^-7
1,2,3,4,7-PentaCDD (1.2±0.1) x 10^-7 (4.6±0.1) x 10^-7
1,2,3,4,7,8-HexaCDD (4.4±0.1) x 10^-9 (19.0±0.1) x 10^-9
1,2,3,4,6,7,8-HeptaCDD (2.4±0.3) x 10^-9 (6.3±0.2) x 10^-9
OctaCDD (0.4±0.1) x 10^-9 (2.0±0.2) x 10^-9

^a From: Friesen et al. (1985).

Marple et al. (1986a) have reanalysed the water solubility of
2,3,7,8-TCDD and found it to be considerably less (12.5-19.2
ng/litre). The log water-octanol partition coefficient (K_ow) has
been determined as 6.64 by Marple et al. (1986b).

Friesen et al. (1985) have determined the water solubility for
some PCDDs other than the 2,3,7,8-TCDD compound and these are given in
Table 6.

Similarly Webster et al. (1985) have determined the log
octanol-water partition coefficients for a number of PCDDs (Table 7).

2,3,7,8-TetraCDD is considered to be a stable compound, but due
to its extreme toxicity its chemistry has not been fully evaluated.
However, it undergoes substitution reactions (Baughman, 1974) as well
as photochemical dechlorination (Crosby et al., 1971; Crosby & Wong,
1977; Gebefugi et al., 1977). Thermally it is very stable and rapid
decomposition of 2,3,7,8-tetraCDD occurs only at temperatures above
750 °C (Stehl et al., 1973). The other PCDDs have been much less
studied; however, octaCDD is completely destroyed by treatment with
hot alkali (Albro & Corbett, 1977).

The first synthesis of 2,3,7,8-tetraCDD was reported by
Sandermann et al. (1957), who used catalytic chlorination of the
unchlorinated dioxin. It has also been prepared in good yields by the
dimerization of 2,4,5-trichlorophenol salts (Buu-Hoi et al., 1971b;
Langer et al., 1973).

In the PCDF series, Mazer et al. (1983) synthesized all the 38
positional tetraCDF isomers. The products were mixtures of isomers,
and each of these isomers could be identified. Later Bell & Gari,
(1985) isolated and characterized all the 38 tetraCDFs, 28 pentaCDFs,
and 16 hexaCDFs.

Table 7. Values for log K_ow for some PCDDs from linear and quadratic plots

log K_ow (linear) log K_ow (quadratic)

Waters Waters Waters Waters
Bondapak Bondapak Bondapak Bondapak
Compound (Woodburn data) (Woodburn data)

Dibenzo-p-dioxin 4.26 4.01 4.34 4.17
1-MonoCDD 4.81 4.52 4.91 4.75
2-MonoCDD 5.33 5.00 5.45 5.29
2,7-DiCDD 6.27 5.86 6.39 6.17
1,2,4-TriCDD 7.36 6.86 7.45 7.11
1,2,3,7-TetraCDD 8.15 7.58 8.19 7.72
1,2,3,4-TetraCDD 8.63 8.02 8.64 8.07
1,3,6,8-TetraCDD 8.70 8.08 8.70 8.12
1,2,3,4,7-PentaCDD 9.48 8.80 9.40 8.64
1,2,3,4,7,8-HexaCDD 10.40 9.65 10.22 9.19
1,2,3,4,6,7,8-HeptaCDD 11.38 10.55 11.05 9.69
OctaCDD 12.26 11.35 11.76 10.07

a From: Webster et al. (1985).

Kuroki et al. (1984) have synthesized 51 congeners of PCDFs by a
structure specific method from chlorophenols and chloronitrobenzenes
or chlorophenols and chlorodiphenyls iodonium salts. The structures
were confirmed by MS and NMR.

Safe & Safe (1984) described the synthesis of 22 PCDF congeners
resulting in quantities of 10-320 mg of purified product. They also
reported NMR data on the compounds synthesized.

Sarna et al. (1984) and Burkhard & Kuehl, (1986) have documented
the octanol/water partition coefficients for some PCDFs (Table 8). The
disagreement for OCDF arises because of uncertainties in the K_ow
values of reference compounds of high K_ow. The partitioning of
organic chemicals between lipid and water is an important determinant
of the bioconcentration potential of a toxicant and has sometimes been
effectively used as an indicator of the preferred degradative in in
vivo pathways.

Table 8. The logarithm of the octanol/water partition coefficients (K_ow) of some PCDFs using HPLC methods

PCDF log K_ow Reference

2,8-dichloro- 5.95 Sarna et al. 1984^a
5.30^b Burkhard & Kuehl, 1986^c

2,3,7,8-tetrachloro- 5.82±0.02 Burkhard & Kuehl, 1986^c

octachloro- 13.37 Sarna et al. 1984^a
8.78 Burkhard & Kuehl, 1986^c

^a Quadratic equation treatment: Biorad Biosil (10 mm) data.
^b Quadratic equation treatment: unspecified "microbore" HPLC column.
^c Sarna et al. (1984) data recalculated from experimental data.

2.3. Analytical Methods

2.3.1 General aspects

The earliest reported method used to detect 2,3,7,8-tetraCDD was
a rabbit skin test (Adams et al., 1941). Test samples were applied to
the inner surface of the ear and to the shaven belly of albino
rabbits, and inflammatory responses were observed. Subsequently, Jones
& Krizek (1962) developed a test based on the recovery and weight of
the keratin formed on the rabbit ear after application of a sample.
These biological methods were non-specific as to isomers and not
sufficiently sensitive to detect low levels of contamination.

In the late 1960s and early 1970s, gas chromatographic methods
were used for the quantification mainly of 2,3,7,8-tetraCDD in
commercial 2,4,5-T formations. The detection level was normally in the
range of µg/g. These analyses were not isomer-specific and the results
could not be confirmed. Ryhage (1964) solved the problem of combining
a gas chromatograph with a mass spectrometer. During the 1970s and
1980s, various types of mass spectrometer and gas chromatograph/mass
spectrometer combinations were used in analytical work. Use of these
more sophisticated instruments allowed for the development of
isomer-specific and validated analyses for the tetraCDDs in the very
late 1970s and for the other PCDDs and PCDFs in the early 1980s.

A number of spectroscopic methods are available for the
laboratory identification of 2,3,7,8-tetraCDD, but their use is highly
restricted, with the exception of mass spectroscopy (MS). Data on
X-ray, infra-red (IR), ultra-violet (UV), nuclear magnetic resonance
(NMR), electron spin resonance (ESR), and mass spectra were obtained
by Pohland & Yang (1972), Baughman (1974), and Slonecker et al.
(1983).

Because of the large number of isomers and congeners, and due to
the extreme toxicity of some PCDD and PCDF isomers, highly sensitive
and specific analytical techniques are required for the measurements.
Detection limits for the analysis of environmental and human samples
should be orders of magnitude lower than the usual detection levels
required for pesticide analysis. A detection level of 1 pg or less
might be required to measure 2,3,7,8-tetraCDD and the other toxic
isomers in a 1-g environmental sample. Analyses at such low levels are
complicated by the presence of a multitude of other interfering
compounds and clean-up procedures are required.

The mono-, di-, and trichloro congeners are not usually included
in these analyses. Such compounds are considered to be much less toxic
than the higher chlorinated congeners and are also much more volatile
and losses may occur during clean-up.

It should be mentioned that the level of sophistication needed in
the analyses for PCDD and PCDFs will depend upon the objectives
thereof. In cases where the objectives were primarily to screen
samples to identify groups of PCDDs and/or PCDFs (in a qualitative or
semiquantitative manner), routine assays and bioassays were adequate.
In other instances, where the objective of the analysis was to
quantify accurately specific PCDD and/or PCDF isomers in the samples,
sophisticated analytical procedures were required. Clearly, both types
of analyses can be useful, depending on the purpose for which the
analytical results are to be used.

Many analytical methods have been developed in recent years for
the analysis of trace amounts of PCDDs and PCDFs in environmental
samples, especially for 2,3,7,8-tetraCDD. The most specific of these
methods are based on MS. There are many requirements to be met by such
an analytical method, including representative sampling and
appropriate storage, efficient extraction, high selectivity in the
clean-up, high specificity in the gas chromatography, high sensitivity
in the detection, safe and reliable quantification, good
reproducibility, useful confirmatory information.

Several review articles discussing methods of analyzing PCDDs and
PCDFs have appeared (McKinney, 1978; Esposito et al., 1980; Rappe &
Buser, 1980; Harless & Lewis, 1982; Karasek & Anuska, 1982; Tiernan,
1983; Crummett et al., 1985). Most of the older methods have been
critically reviewed by a panel of experts assembled by the National
Research Council of Canada (1981).

2.3.2 Sampling strategy and sampling methods

The quality and utility of analytical data depend on the validity
of the sample and the adequacy of the sampling program. The purpose of
sampling is to obtain specimens that represent the situation being
studied. Sampling plans may require that systematic samples be
obtained at specified times and places, or simple random sampling may
be necessary. Generally, the sample should be an unbiased
representation of the environmental situation.

All aspects of a sampling programme should be planned and
documented in detail, and the expected relationship of the sampling
protocol to the analytical result should be defined. A sampling
programme should include reasons for choosing sampling sites, the
number and type of samples, the timing of sample acquisition and the
sampling equipment used. A detailed sampling procedure should include
a description of the sampling situation, the sampling methodology,
labelling of samples, field blank preparation, pretreatment
procedures, and transportation and storage procedures.

The quality assurance programme should include means to
demonstrate that containers or storage procedures do not alter the
qualitative or quantitative composition of the sample. Special
transportation and storage procedures (refrigeration or exclusion of
light) should be described, if they are required.

Because environmental samples are typically heterogeneous, a
sufficiently large number of samples (ten or more) must normally be
analyzed to obtain meaningful data on chemical composition. The number
of individual samples that should be analyzed will depend on the kind
of information required by the investigation. If an average
compositional value is required, a number of randomly selected
individual samples may be obtained, combined, and blended to provide
a homogeneous composite sample from which a sufficient number of
subsamples could be analyzed. If composition profiles, time trends, or
the variability of the sample population are of interest, many samples
need to be collected and analyzed individually.

If field blanks are not available, efforts should be made to
obtain blank samples that best simulate a sample that does not contain
the specific chemical. In addition, measurements should be made to
ascertain whether, and to what extent, any reagent or solvent used may
contribute to or interfere with the analytical results (laboratory and
solvent blanks).

The recovery tests are frequently used and necessary to evaluate
the analytical methodology. Uncontaminated samples from control sites
that have been spiked with the chemical of interest provide the best
information because they simulate any matrix effect. When feasible,
isotopically labelled (¹³C, ³⁷Cl) chemicals spiked into the sample
provide the greatest accuracy since they are subjected to the same
matrix effects. The ¹³C- and ³⁷Cl-labelled compounds can be used
to validate:

(a) sampling (sampling surrogate),
(b) analytical pretreatment (clean-up surrogate),
(c) quantification (internal standard).

Very few laboratories in the world have access to and experience
in working with these complicated analyses.

In order to be able to compare data generated in different
laboratories, the same quantitative standard compounds should be used.
Interlaboratory calibrations or "round-robin" studies have been
performed in very few cases.

2.3.3 Extraction procedures

In this step, the sample is homogenized or digested and extracted
with a suitable solvent or solvent mixture to remove the bulk of the
sample matrix and transfer the PCDD and PCDF residue into the solvent.
Both the selection of the proper solvent and the method of extraction
can be critical in obtaining a satisfactory recovery of PCDDs and
PCDFs from the sample matrix.

Many different procedures for the extraction of PCDDs/PCDFs from
various samples are described. In some cases this involves digestion
or destruction of the matrix. Some of these methods have been
evaluated in the report from the National Research Council of Canada
(1982), while other methods are discussed by Tiernan (1983).

An interlaboratory "round-robin" study involving 13 laboratories
was carried out to evaluate the reliability of data on
2,3,7,8-tetraCDD in fish samples. No significant differences were
found from methods differing in the digestion or extraction procedures
(Ryan et al., 1983b).

In a study described by Albro et al. (1985), eight different
approaches were applied in eight laboratories to quantify four PCDDs
(2,3,7,8-tetraCDD; 1,2,3,7,8-pentaCDD; 1,2,3,4,7,8-hexaCDD; and
octaCDD) and three PCDFs (2,3,7,8-tetraCDF; 2,3,4,7,8-pentaCDF; and
1,2,3,7,8,9-hexaCDF) in spiked samples of an extract from human
adipose tissue. Levels of fortification, unknown to the participating
laboratories, were in the 5-50 ng/kg range, except for octaCDD (up to

500 ng/kg). The results indicated that most of the procedures tested
gave a high degree of qualitative reliability. However, other methods
were not so accurate, a large portion of the reported data consisting
of false positives or false negatives.

Lustenhouwer et al. (1980) studied the extraction of PCDDs and
PCDFs from a fly ash sample. A dramatic difference was found between
different solvents.

2.3.4 Sample clean-up

In the sample clean-up, the PCDDs and PCDFs present in the sample
should be separated from a multitude of other co-extracted and
possibly interfering compounds. The clean-up methods, normally three
steps or more, can vary for different sample matrices. Two different
procedural trends can be recognized:

(a) all PCDD and PCDF isomers can be analyzed in one
single fraction by the containment enrichment
procedure (Norstrom et al., 1982; Stalling et al.,
1983; Tiernan, 1983; Rappe, 1984),

(b) specific isomers are analyzed in different fractions
mainly after normal-phase and reverse-phase high
pressure liquid chromatography (HPLC) separation
(Lamparski et al., 1979; Niemann et al., 1983; Tosine
et al., 1983).

This latter method allows the identification of only a few PCDD
isomers in each fraction, and is mainly used to monitor TCDD and a few
other congeners. For a monitoring program of all PCDDs and PCDFs a
more general method might be preferred.

The method described by Stalling et al. (1983) was originally
designed for the analyses of fish samples. In a "round-robin" study of
fish samples it gave good results (Ryan et al., 1983b). This method
has now been used for the clean-up of other biological samples like
bird muscle, seal fat, turtle fat, and human adipose tissue - blood,
liver, kidney, and milk (Rappe et al., 1983c; Nygren et al., 1986;
Rappe et al., 1986b).

2.3.5 Isomer identification

The purified extracts are used directly for the final analyses
with the aid of a gas-chromatograph/mass spectrometer (GC/MS) equipped
with a glass capillary or a fused-silica column. The column leads
directly into the ion source of the mass spectrometer, which operates
either in the electron impact (EI) or the negative ion-chemical
ionization (NCI) mode. In view of the large variation in toxicological

and biological effects of the PCDD and PCDF isomers, it is imperative
that the isomers, particularly those having high toxicity, be
identified. For an unambiguous isomer identification it is necessary
to have access to all analytical standards within a specific group of
isomers, e.g. all the 22 tetraCDDs and all the 38 tetraCDFs. All the
22 tetraCDDs have been prepared and, using a Silar 10c glass capillary
column, the 2,3,7,8-tetraCDD can be separated from all the other 21
tetra isomers (Buser & Rappe, 1980). Recently all the 14 pentaCDDs and
the 10 hexaCDDs have been prepared. Using the Silar 10c column all the
2,3,7,8- substituted isomers can be separated from all the other
isomers (Buser & Rappe, 1984). The SP 2330 fused silica column can
also be used for this separation (Rappe, 1984).

In the PCDF series, Mazer et al. (1983) have synthesized all the
38 positional tetraCDF isomers. The products were mixtures of isomers,
and each of these isomers could be identified using both an SP 2330
and an SE 54 capillary column. Later, Bell & Gara (1985) isolated and
characterized all tetra-, penta- and hexaCDFs. The SP 2330 column can
separate most of these isomers (Rappe, 1984). The 1,2,3,7,8-pentaCDF
co-elutes with the 1,2,3,4,8-isomer and the 1,2,3,4,7,8- hexaCDF with
the 1,2,3,4,7,9-isomer, but they can be separated on less polar
columns like OV-17 and DB-5.

A very limited number of investigations has been performed using
these complete sets of synthetic standards.

2.3.6 Quantification

Mass selective detection (mass fragmentography) has been used to
quantify trace amounts of PCDDs and PCDFs in the samples by
selectively monitoring M, M + 2, and/or M + 4 ions (SIM). The
quantification is based on peak area measurements and a comparison of
these areas using either isotopically labelled internal standards
(¹³C or ³⁷Cl) or calibration curves of external standards. As a
first approach, it has been generally assumed that with the MS
quantification technique, all isomers of a particular congener of PCDD
or PCDF (e.g. the tetrachloro-isomers) have the same response factors.
However, an investigation of 13 well-defined tetraCDF isomers has
shown a three-fold variation in response factors with the EI mode and
up to a 20-fold variation with the negative ion-chemical ionization
mode. For the higher chlorinated homologues (penta, hexa) the
variation was found to be less (Rappe et al., 1983b).

Fung et al. (1985) have studied the mass spectra of 26 PCDF
congeners. They found that the EI spectra are not particularly isomer
specific, while positive ion-chemical ionization spectra show a
greater degree of isomer distinction.

2.3.7 Confirmation

Quality control and quality assurance programs help to assure
that positive data reported actually refer to specific PCDDs and PCDFs
(Kloepfer et al., 1983). To provide reliable data:

(a) isomer specificity must be demonstrated initially and verified
daily,
(b) the retention time must equal (within 3 seconds) the retention
time for the isotopically labelled congener,
(c) the signal to noise ratio must be 2.5:1 or higher,
(d) the chlorine cluster must be within ± 10% of the theoretical
values, given in Table 9,
(e) correct fragments, e.g., M⁺-COCl ions, must be with correct
chlorine clusters.

For confirmation, mass spectroscopy is the best technique now
available. The EI mass spectral properties of PCDFs and PCDD have been
described (Buser, 1975). The molecular (M⁺) and fragment ions of
PCDDs and PCDFs show the typical, expected clustering due to the
chlorine isotopes (Table 9). The typical fragmentation is M^-COCl⁺,
which is a useful fragment to study.

Buser & Rappe (1978) have shown that observation of low mass ions
can be used for the identification of the substitution pattern of
PCDDs, which can be defined as the number of chlorine atoms on each
carbon ring of the dioxin molecular; the 2,3,7,8-isomer has a 2:2
pattern while 1,2,3,4-tetraCDD has a 4:0 pattern. However, these low
mass ions may not be observed in spectra from environmental or
biological samples.

In the negative ion-chemical ionization mode, the PCDFs have the
base peak due to M^-, and the fragmentation produces the unusual
M^--34 ions (uptake of H and loss of Cl). Fragmentation of PCDDs in
this mode is more conventional via loss of Cl yielding M^--35 ions
(Buser et al., 1985).

Using EI technique and a quadropole instrument, the detection
limits are 1-10 pg for the tetrachloro compounds and up to 10-50 pg
for the octachloro compounds using selected ion monitoring or multiple
ion detection (SIM or MID). Full mass spectra require 0.1-1 ng of
compound (Buser et al., 1985). High resolution instruments can improve
the sensitivity by one order of magnitude.

The negative ion-chemical ionization mode, using methane gas as
reagent, gas provides extremely good sensitivity for all PCDFs (tetra-
to octachloro- compounds) and for the higher chlorinated PCDDs (penta-
to octaCDD). The detection limits are in the 10-100 fg (10^-15g)
range using SIR or MID, which is 1 to 2 orders of magnitude better
than EI (Buser et al., 1985). However, the negative ion-chemical
ionization mode has very poor sensitivity for 2,3,7,8- tetraCDD under
these conditions.

Using low resolution MS instruments, a series of interfering
compounds has been identified (Table 10). Some of this interference
can be eliminated using high resolution MS instruments operating at
8000 - 10 000 daltons. However, compounds with the same empirical
formulae cannot be separated by MS technique; they are normally
eliminated during the clean-up or separated by the gas chromatography
step.

2.3.8 Other analytical methods

Paasivirta et al. (1977) have shown that 2,3,7,8-tetraCDD can be
detected down to the pg level using a glass capillary column and a
⁶³Ni electron-capture detector. Combined with efficient clean-up
procedures, this method has shown to be useful down to a level of 9
ppt (Niemann et al., 1983), although positive samples need
confirmation by mass spectroscopy (MID, SIM).

Other techniques, such as enzyme induction and radioimmunoassay
have been described and discussed by Firestone (1978) and McKinney
(1978). McKinney et al. (1982) have used the radioimmunoassay method
for determining 2,3,7,8-tetraCDD in human fat, and found the reliable
sensitivity at 95% confidence interval to be 100 pg per sample.

An analytical method based on the keratonization response of
epithelial cells in an in vitro system has been described by
Gierthy & Crane (1985b). This method can be an assay for dioxin-like
activity in environmental and biological samples. A positive response
was found for 2,3,7,8-tetraCDD at a concentration of 10-11 mol/litre.

Table 9. Isotopic abundance ratio ("cluster") of polychlorinated dioxins and dibenzofurans

Number of
chlorine M M + 2 M + 4 M + 6 M + 8 M + 10 M + 12 M + 14
atoms

1 100.0 33.7
2 100.0 66.1 11.3
3 100.0 98.4 32.7 3.8
4 76.4 100.0 49.4 11.0 1.0
5 61.2 100.0 65.5 21.6 3.6 0.3
6 51.1 100.0 81.7 35.8 8.9 1.2 0.1
7 43.8 100.0 97.9 53.4 17.6 3.5 0.4
8 33.7 87.6 100.0 65.3 26.8 7.0 1.2 0.1

Table 10. List of molecular ions of polychlorinated compounds present in some human and environmental samples
and possibly interfering in the mass spectral analysis of PCDFs and PCDDs^a

Molecular ions (m/z,m⁺,m^-) (chlorination)
Compounds mono- di- tri- tetra- penta- hexa- hepta- octa- nona- deca-

PCDDs 320 354 388 422 456 - -
PCDFs 304 338 372 406 440 - -

PCBs 290 324 358 392 426 460 494
PCNs 264 298 332 366 400 - -
PCTs 298 332 366 400 434 468 502 536 570
PCDPEs^b 238 272 306 340 374 408 442 476 510
PCPYs^c 36 270 304 338 372 406

^a From: Buser et al. (1985).
^b PCDPEs: Polychlorinated diphenylethers.
^c PCPYs: Polychlorinated pyrenes.

3. SOURCES OF ENVIRONMENTAL POLLUTION

3.1 Production, Synthesis, and Use

PCDDs and PCDFs are not produced commercially. These compounds
are in fact formed as trace amounts of undesired impurities in the
manufacture of other chemicals such as chlorinated phenols and their
derivatives, chlorinated diphenyl ethers, and polychlorinated
biphenyls (PCBs). There is no known technical use for the PCDDs and
PCDFs.

The amount of total PCDDs entering the Canadian environment/year
has been estimated to be about 1500 kg, and 75% of this amount has
been estimated to be due to octaCDD alone (National Research Council
of Canada, 1981). There is no estimation of the amount of PCDFs
entering the environment anywhere in the world.

Although the polychlorinated dioxins and dibenzofurans are not
commercially produced, most of these compounds have been synthesized
for research purposes in small quantities according to the reactions
discussed in section 2.

3.2 Industrial Processes

In addition to the synthetic methods mentioned in section 2,
2,3,7,8-tetraCDD may be formed during the industrial preparation of
2,4,5-trichlorophenol from 1,2,4,5-tetra-chlorobenzene. This
substitution reaction takes place at about 180 °C, and when the
solvent is methanol, the pressure rises to about 7 KPa. The formation
of TCDD is an unwanted side reaction which takes place when the
reaction mixture is heated to 230-260 °C (Milnes, 1971). This reaction
is exothermic, so that even higher temperatures may be attained
resulting in uncontrolled conditions.

In some factories ethylene glycol is used as a solvent in order
to avoid the high pressure. As already pointed out by Milnes (1971),
however, use of this solvent requires special precautions because of
the occurrence of a base-promoted polymerization of ethylene glycol
and decomposition reactions that produce ethylene oxide. These
reactions are also exothermic; they may start spontaneously at
temperatures above 180 °C and proceed rapidly and uncontrollably to
result in the formation of relatively large amounts of TCDD.

After most of the solvent has been removed, the reaction mixture
is acidified; the 2,4,5-trichlorophenol can be separated from
2,3,7,8-tetraCDD by one or two distillations, with the result that
2,3,7,8-tetraCDD is concentrated in the still-bottom residues. Up to
1 mg/g of 2,3,7,8-tetraCDD in such residues has been reported
(Kimbrough et al., 1984). Improper disposal of such residues is
discussed in sections 4.4.2 and 9.

Most of the 2,4,5-trichlorophenol produced is used for the
preparation of herbicides such as 2,4,5-T (including various esters
and salts, and the bactericide hexachlorophene).

PCDDs and PCDFs are both formed as by-products during the
manufacture of chlorinated phenols (2,4-dichloro-, 2,4,6-trichloro-,
2,3,4,6-tetrachloro- and pentachlorophenol). The commercial
chlorophenols are produced by two processes, i.e., by chlorination of
the phenol using various catalysts and by the alkaline hydrolysis of
an appropriate chlorobenzene. Apparently both reactions can lead to
the formation of PCDDs as well as PCDFs, and the level of
contamination is normally much higher here than in the production of
2,4,5-trichloro-phenol (see section 3.3).

PCDDs and PCDFs are also formed during the preparation of
chlorinated diphenyl ether herbicides (Yamagishi et al., 1981) and
hexachlorobenzene (Villeneuve et al., 1974). A series of PCDFs are
formed during the production of PCBs (see section 3.3).

Production equipment is often used for the production of several
different chemicals. In the manufacture of chemicals on such equipment
previously contaminated by PCDDs and PCDFs, both the products and
waste generated can be contaminated. Thus, manufactured
2,4-dichlorophenoxyacetic esters (2,4-D), which otherwise should not
be contaminated by 2,3,7,8-tetraCDD, did indeed contain this dioxin
because the equipment used had been employed previously to produce
2,4,5-T and had not been cleaned properly (Federal Register, 1980).

It should be pointed out that the primary occurrence of TCDD in
the environment is possibly related to the synthesis of
2,4,5-trichlorophenol, the use of products prepared from this compound
(Table 11), and to incinerations reactions. The occurrence of the
other PCDDs and PCDFs is related to the synthesis and use of a variety
of other products (Table 12), some of which are quite common.

The other PCDDs and PCDFs are also formed in a variety of
incineration reactions (see section 4.5).

3.3 Contamination of Commercial Products

3.3.1 Chlorophenoxyacetic acid herbicides

Depending on the temperature control and purification efficiency,
the levels of 2,3,7,8-tetraCDD in commercial products may vary
greatly. For example, the levels of 2,3,7,8-tetraCDD in drums of the
herbicide Agent Orange placed in storage in the USA and in the Pacific

before 1970 were between 0.02 and 47 mg/g. More than 450 samples were
analyzed in this study, and the mean value was 1.98 mg/g (Young et
al., 1983). Since Agent Orange was formulated as a 1:1 mixture of the
butyl esters of 2,4,5-T and 2,4-D, the levels of 2,3,7,8-tetraCDD in
individual 2,4,5-T preparations manufactured and used in the 1960s
could have been as high as 100 mg/g.

In analyses using high-resolution GC-MS, Rappe et al. (1978a)
have reported that in other samples of Agent Orange (as well as in
European and the USA 2,4,5-T formulations from the 1950s and 1960s),
2,3,7,8-tetraCDD was the dominating compound of this group of
contaminants. Only minor amounts of other PCDDs and PCDFs could be
found, primarily lower chlorinated PCDDs, in samples of Agent Orange.

As a result of governmental regulations, efforts were made during
the 1970s to minimize the formation of 2,3,7,8-tetraCDD during 2,4,5-T
production, and now all producers claim that their products contain
less than 0.1 µg 2,3,7,8-tetraCDD/g of product (Rappe et al., 1978a).
At present, the chloro-phenoxy herbicides are not the major source of
PCDDs and PCDFs in the environment.

Sixteen samples of 2,4-D esters and amine salts from Canada were
analyzed for the presence of PCDDs. Eight out of nine esters and four
out of seven amine salts were found to be contaminated, with the
esters showing significantly higher levels (210-1752 ng/g) than the
salts (20-278 ng/g). The tetraCDD observed was the 1,3,6,8-isomer, as
verified by a synthetically prepared authentic standard (Cochrane et
al., 1981). In other studies, it has been found that no tetraCDD other
than the 1,3,6,8-isomer elutes in this window. Hagenmaier et al.
(1986) has reported that, unexpectedly, a German 2,4-D formulation
contained 6.8 ng of 2,3,7,8-tetraCDD/g.

Table 11. Some commercial products that may be contaminated with
2,3,7,8-tetraCDD, depending on the method of preparation

Common name Chemical name

2,4,5-T^a 2,4,5-Trichlorophenoxyacetic acid

2,4,5-T esters^a n-butyl-, butoxy ethyl-, and
iso-octyl-esters of 2,4,5-
trichlorophenoxyacetic acid

2,4,5-T salts^a dimethylamine salts of 2,4,5-
trichlorophenoxyacetic acid

Fenoprop esters of 2-(2,4,5-trichlorophenoxy)-
propanoic acid

Erbon ethyl ester of 2-(2,4,5-trichloro-
phenoxy)-2,2-dichloropropanoic acid

2,4,5-Trichlorophenol 2,4,5-Trichlorophenol
Fenochlorphos O,O-Dimethyl O-2,4,5-trichlorophenyl
phosphonothioate

Trichloronate O-Ethyl 0-2,4,5-trichlorophenyl
ethylphosphonothioate

Hexachlorophene/isobac 20 2,2'-Methylene-bis (3,4,6-trichloro-
phenol)

^a There are numerous trade names for this product.

Table 12. Some commercial products which may be contaminated with PCDDs
other than 2,3,7,8-tetraCDD, and with PCDFs, depending on the method of
preparation

Common name Chemical name

Bifenox Methyl-5-2,4-dichlorophenoxy-2-nitrobenzoate

Chloranil 2,3,5,6-Tetrachloro-2,
5-cyclo-hexadiene-1,4-dione.

2,4-D (esters and salts) 2,4-Dichlorophenoxyacetic acid
and esters and salts

2,4-DB and salts 2,4-Dichlorophenoxybutyric acid and
salts

Dicamba 3,6-Dichloro-2-methoxybenzoic acid

Dicamba, dimethylamine salt 3,6-Dichloro-2-methoxybenzoic acid,
dimethylamine salt

Dicapthon Phosphorothioic acid
o-(2-chloro-4-nitrophenyl)
o,o-dimethyl ester

Dichlofenthion Phosphorothioic acid
o-2,4-dichloro-phenyl
o,o-dialkyl ester

Disul sodium (sesone) 2,4-Dichlorophenoxyethyl sulfate,
sodium salt

2,4-DP 2- 2,4-Dichlorophenoxy propionic acid

HCB Hexachlorobenzene

Nitrofen 2,4-Dichlorophenyl-p-nitrophenyl
ether

PCP and salts Pentachlorophenol and salts

PCB Polychlorinated biphenyls

2,4,6-TCP 2,4,6-Trichlorophenol and salts
2,3,4,6-Tetrachlorophenol and salts

Common name Chemical name

CNP 1,3,5-Trichloro-2-(4-nitrophenoxy)
benzene

NIP 2,4-Dichloro-1-(4-nitrophenoxy)
benzene

X-52 2,4-Dichloro-1-(3-methoxy-4-nitro-
phenoxy) benzene

3.3.2 Hexachlorophene

The bactericide hexachlorophene is prepared from
2,4,5-trichlorophenol, also the key intermediate in the production of
2,4,5-T. Due to additional purification, the level of 2,3,7,8-tetraCDD
in this product is usually < 0.03 mg/kg (Baughman, 1974). Ligon & May
(1986) reported 0.0047 mg/kg of TCDD in one hexachlorophene sample.
However, hexachlorophene also contains about 100 mg/kg of a
hexachloroxanthene, the 1,2,4,6,8,9-substituted isomer (Göthe &
Wachtmeister, 1972).

3.3.3 Chlorophenols

Chlorophenols have been used extensively since the 1950s as
insecticides, fungicides, mold inhibitors, antiseptics, and
disinfectants. In 1978 the annual world production was estimated to be
approximately 200 000 tons. The most important use of 2,4,6-tri-,
2,3,4,6-tetra-, and pentachlorophenol, and their salts, is for wood
preservation. Pentachlorophenol is also used as a fungicide for slime
control in the manufacture of paper pulp and for a variety of other
purposes such as in cutting oils and fluids, for tanning leather, and
in paint, glues, and outdoor textiles. 2,4-Di- and
2,4,5-trichloro-phenol are used for the production of 2,4-D and
2,4,5-T herbicides (phenoxy acids), and 2,4,5-trichlorophenol for the
production of hexachlorophene.

Chlorophenols are produced industrially either by direct
chlorination of phenol or by hydrolysis of chlorobenzenes, the actual
process used depending on the isomer desired. Chlorination of phenol
yields 2,4-di-, 2,4,6-tri-, 2,3,4,6-tetra-, or pentachlorophenol,
while hydrolysis of chlorobenzenes is mainly used for the production
of 2,4,5-tri- and pentachlorophenol (Nilsson et al., 1978).
Chlorophenols may contain a variety of by-products and contaminants,
such as other chlorophenols, polychlorinated phenoxyphenols, and
neutral compounds like polychlorinated benzene and diphenyl ethers

(PCDPEs), PCDDs, and PCDFs. Some of these contaminants may also occur
in chlorophenol derivatives like phenoxy acids, other pesticides, and
hexachlorophene. The possible presence of PCDDs and PCDFs in
commercial products is of special significance because of their
extraordinary persistence and toxicological properties (see sections
7-9). A scientific criteria document for chlorophenols and their
impurities in the Canadian environment has been prepared by Jones
(1981, 1984). Chlorophenols were estimated to be the major chemical
sources of PCDDs and PCDFs in the Canadian environment (Sheffield,
1985).

Buser & Bosshardt (1976) reported on the results of a survey of
the PCDD and PCDF contents of pentachlorophenol (PCP) and PCP-Na from
commercial sources in Switzerland. From the results, a grouping of the
samples into two series can be observed: a first series with generally
low levels (hexaCDD <1 µg/g) and a second series with much higher
levels (hexaCDD >1 µg/g) of PCDDs and PCDFs. Samples with high PCDD
values had also high PCDF values. For most samples, the contents of
the PCDF contaminants were in the order:

tetra- = penta- < hexa- < hepta- < octaCDD/CDF.

The ranges of the combined levels of PCDDs and PCDFs were 2-16 and
1-26 µg/g, respectively, for the first series of samples, and 120-500
and 85-570 µg/g, respectively, for the second series of samples. The
levels of octaCDD and octaCDF were as high as 370 and 300 µg/g,
respectively.

Some PCP-Na samples analyzed showed the unexpected presence of a
tetraCDD (0.05-0.25 µg/g), which was later identified by Buser & Rappe
(1978) as the unusual 1,2,3,4-substituted isomer. Table 13 collects a
number of relevant analyses of these chlorophenol formulations. The
levels of PCDDs and PCDFs are higher than for the phenoxy-acetic acid
herbicides.

It has also been reported that several positional isomers of
PCDDs and PCDFs are present in the chlorophenols. However,
isomer-specific methods have not been used in most of these
investigations, and more research is necessary to identify all the
isomers present for a risk evaluation of these products.

Miles et al. (1985) have analyzed PCP samples for hexaCDDs from
five different manufacturers using an isomer-specific analytical
method. The study included both free PCPs as well as the sodium salts.
Total hexaCDDs in PCPs ranged from 0.66 to 38.5 mg/kg, while in the
sodium salts levels of hexaCDDs between 1.55 and 16.3 mg/kg were
found. The most abundant hexaCDD isomer found in the free PCPs was the
1,2,3,6,7,8 isomer; however, in the sodium salts the 1,2,3,6,7,9- and
1,2,3,6,8,9-hexaCDD pair was the most abundant.

Table 13. Levels of PCDDs and PCDFs in commercial chlorophenols (µg/g)^a

2,4,6- 2,3,4,6- PCP PCP
Trichlorophenol Tetrachlorophenol Sample A Sample B

TetraCDDs < 0.1 < 0.1 < 0.1 < 0.1
PentaCDDs < 0.1 < 0.1 < 0.1 < 0.1
HexaCDDs < 1 < 1 < 1 2.5
HeptaCDDs < 1 10 0.5 175
OctaCDD < 1 2 4.3 500
TetraCDFs 1.5 0.5 < 0.1 < 0.1
PentaCDFs 17.5 10 < 0.1 < 0.1
HexaCDFs 36 70 0.03 < 0.3
HeptaCDFs 4.8 70 0.5 19
OctaCDF < 1 10 1.1 25

^a From: Rappe et al. (1979).

Hagenmaier & Brunner (1987) has reported that 2,3,7,8-tetraCDD
can be found in commercial pentachlorphenol formulation at levels of
0.21-0.56 ng/g, while Hagenmeyer & Brunner (1986) report that
1,2,3,7,8-pentaCDD was found in pentachlorophenol and
Na-pentachlorophenates in concentrations of 0.9-18 ng/g.

3.3.4 Polychlorinated biphenyls (PCBs)

Vos et al. (1970) were able to identify PCDFs (tetra- and
pentaCDFs) in samples of European PCBs (Phenoclor DP-6 and Clophen A
60) but not in a sample of Aroclor 1260. The toxic effects of these
PCB products were found to parallel the levels of PCDFs present. Bowes
et al. (1975) examined a series of Aroclors, as well as the samples of
Aroclor 1260, Phenoclor DP-6, and Clophen A-60 previously analyzed by
Vos et al. (1970). They used packed columns and very few standard
compounds, and reported that the most abundant PCDFs had the same
retention time as 2,3,7,8-tetraCDF and 2,3,4,7,8-pentaCDF. Using a
complete set of PCDF standards and an isomer-specific analytical
method, Rappe et al. (1985d) determined the levels of
2,3,7,8-substituted PCDFs in commercial PCB products (see Table 14).

3.3.5 Chlorodiphenyl ether herbicides

In 1981, Yamagishi et al. reported on the occurrence of PCDDs and
PCDFs in the commercial diphenyl ether herbicides
1,3,5-trichloro-2-(4-nitrophenoxy) benzene (CNP),
2,4-di-chloro-1-(4-nitrophenoxy)benzene (NIP), and
2,4-dichloro-1-(3-methoxy-4-nitrophenoxy)benzene (X-52). The total
tetraCDD found was 14.0 mg/kg in CNP, 0.38 mg/kg in NIP, and 0.03 in
X-52. Very few synthetic standards were used, but the major tetraCDDs
were identified as the 1,3,6,8- and 1,3,7,9-isomers, the expected
impurities in the starting material 2,4,6-trichlorophenol. No
2,3,7,8-tetraCDD could be detected in these samples. In all three
herbicides, total tetraCDF was between 0.3 and 0.4 mg/kg.

3.3.6 Hexachlorobenzene

Hexachlorobenzene was used for the control of wheat bunt and
fungi. Villeneuve et al. (1974), analyzing three commercial
hexachlorobenzene preparations, identified octaCDD and hepta- and
octaCDFs. The levels and identity of the heptaCDF isomers were not
given. Great variation in levels of octaCDDs between the three samples
(0.05-211.9 mg/kg) was noted, as well as in the level of octaCDF
(0.35-58.3 mg/kg).

3.3.7 Rice oil

In 1968 more than 1500 people in southwest Japan were intoxicated
by the consumption of a commercial rice oil accidentally contaminated
by PCBs, PCDFs, and polychlorinated quarterphenyls (Masuda &
Yoshimura, 1982; Masuda et al., 1985). In 1979 a similar episode
occurred in central Taiwan, the number of people involved here
approaching 2000 (Chen et al., 1980, 1981). Both these accidents have
been referred to as Yusho episodes, but now the Taiwan episode has
been renamed Yu-cheng (see section 5.4.4.4).

The total level of PCDFs in the Japanese rice oil was reported to
be 5 µg/g (Nagayama et al., 1976) and 5.6 µg/g (Buser et al., 1978d).
For the rice oil from Taiwan, Chen et al. (1985) reported the PCDFs
levels to be in the range 0.18-1.68 µg/g.

Buser et al. (1978) analyzed the Japanese rice oil using glass
capillary columns. They found about 50-60 PCDF congeners and also
reported that the 2,3,7,8-tetraCDF was the major isomer among the
tetraCDFs. However, it was later shown that in this column system the
2,3,4,8-tetraCDF co-elutes with the 2,3,7,8-isomer, and in fact the
2,3,4,8-isomer was the main constituent in this peak (Chen & Hites,
1983; Masuda et al., 1985). The 2,3,7,8-substituted congeners were
estimated to account for 10-15% of the total amount of PCDFs (Buser et
al., 1978).

Table 14. PCDFs in commercial PCBs (ng/g)^a

TRI- TETRA- PENTA- HEXA- HEPTA-

Total 2378 Total 12348 23478 Total 123479 123678 123789 234678 Total Total
PCB-type 12378 123478

Pyralene 700 53 630 10 T 35 ND ND ND ND ND ND
A1254 63 19 1400 690 490 4000 2500 2100 190 130 10 000 960
A1260 10 13 110 48 56 260 500 120 190 27 1500 1300
A30 500 35 573 14 28 160 50 59 ND ND 220 T
A40 1300 180 2600 96 8 1700 79 68 ND T 310 ND
A50 7400 3300 20 000 760 1100 8000 700 360 18 98 3100 75
A60 770 840 6900 1100 990 8100 1600 330 170 330 6800 2000
T64 47 23 360 97 122 840 520 390 58 41 2600 220
Clophen C 710 54 1200 34 30 270 ND T ND ND T ND

^a From: Rappe et al. (1985d).
T = traces.
ND = not detected.

3.4 Sources of Heavy Environmental Pollution

3.4.1 Industrial accidents

Several industrial accidents occurring during the production of
2,4,5-trichlorophenol have been described in the literature. In most
of these accidents the pollution of 2,3,7,8-tetraCDD has been to
factories with circumscribed occupational exposure (section 9).
However, on 10 July, 1976, a runaway reaction in a factory at Meda
near Seveso in Northern Italy resulted in the escape of a chemical
cloud of trichlorophenol/phenate containing 2,3,7,8-tetraCDD.

The cloud initially covered an area outside the factory 5 km long
and 700 m wide. On the basis of the TCDD levels found in the
contaminated soil samples, it has been estimated that 2-3 kg of TCDD
was released in this accident. About 80% of this amount was deposited
in an area of 15 ha, within a distance of about 500 m from the plant.
The levels of soil contamination in three zones are given in Table 15
(Pocchiari, 1978).

3.4.2 Improper disposal of industrial waste

In 1973, three horse arenas in Missouri, USA, were found to be
contaminated by high levels of 2,3,7,8-tetraCDD; the highest value
reported was about 30 µg/g of soil (Kimbrough et al., 1977). This
contamination resulted from the application, in 1971, of contaminated
waste oil to control dust at these locations. The TCDD had originated
at a hexachlorophene-producing factory in Verona, Missouri. Additional
tri- and tetraCDDs were also found, but the major component was
1,2,4,6,8,9-hexachloroxanthene, a compound which apparently can serve
as a marker for this type of contamination. The xanthene is a normal
by-product of hexachlorophene production and has never been associated
with the production of 2,4,5-tri-chlorophenol or 2,4,5-T derivatives
(Viswanathan & Kloepfer, 1986).

In 1982, numerous sites of potential 2,3,7,8-tetraCDD
contamination were discovered in eastern Missouri. The contamination
originated from the same waste oil from the factory in Verona. The
streets of the entire town of Times Beach, Missouri, had been sprayed.
More than 10 000 soil samples from Missouri were analyzed. In this
state more than 40 hazardous waste sites containing 2,3,7,8-tetraCDD
were identified. Most of these contaminated sites resulted from the
disposal of waste from the same factory in Verona. The highest level
reported in these soil samples was 9648 mg TCDD/g (Viswanathan &
Kloepfer, 1986).

Another location of great concern is Love Canal, Niagara Falls,
USA. Here, Smith et al. (1983) found high levels of 2,3,7,8-tetraCDD
in storm sewer sediments taken from around the Love Canal waste
disposal site. The highest value was 312 ng/g sediment.

Table 15. Distribution of TCDD contamination in the Seveso area on the basis of soil sample analyses^a

Range Number of soil samples
(µg/m²)

Zone A Zone B Surrounding monitored area

< 0.750 32 25 249
0.750 - 4.99 32 53 128
5.0 - 14.99 6 19 2
15.0 - 49.99 18 6 0
50.0 - 499.99 31 0 0
500.0 - 4999.99 18 0 0
> 5000 3 0 0

^a From: Pocchiari (1978).
Zone A: high-level contamination, about 115 ha.
Zone B: low level contamination, about 255 ha.
Surrounding area: about 1400 ha.

3.4.3 Heavy use of chemicals

The Eglin Air Force Base in Northwest Florida, USA, has been used
for the development and testing of aerial spraying equipment for
military defoliation operations. During the period 1962-1970, a
3-km² test area was sprayed with 73 tons of 2,4,5-T. Analyses of
archived samples of the formulations indicated that approximately 2.8
kg of 2,3,7,8-tetraCDD had been applied as a contaminant of the
herbicide. However, one 37-ha test grid received 2.6 kg of this TCDD
from 1962 to 1964. Levels of 10-1500 ng/kg were found in 22 soil
samples (the top 15 cm) collected and analyzed 14 years after the last
application of herbicide to this site (Young, 1983).

3.5 Other Sources of PCDDs and PCDFs in the Environment

3.5.1 Thermal degradation of technical products

The formation of 2,3,7,8-tetraCDD as a result of thermal
reactions of 2,4,5-T and 2,4,5-T derivatives has been the subject of
controversy. Heating 2,4,5-T salts at 400-450 °C for 30 minutes or
longer yielded approximately 1 g of 2,3,7,8-tetraCDD per kg of 2,4,5-T
salt, while no TCDD was identified from the same treatment of 2,4,5-T
acid or esters (Langer et al., 1973; Baughman, 1974). Using a more
sensitive analytical method, Ahling et al. (1977) reported that 0.2-3
mg of 2,3,7,8-tetraCDD was formed per kg of 2,4,5-T esters during

combustion at 500-850 °C. Two reports (Stehl & Lamparski, 1977;
Andersson et al., 1978) have shown that 2,3,7,8-tetraCDD could not be
found after burning samples of spiked or sprayed vegetation at 600 °C.
The combustion gases, soot, particles, and ashes were analyzed and the
detection limit was 4 mg of TCDD/kg 2,4,5-T burned.

Rappe (1978b) have studied the burning of material impregnated
with various salts of chlorophenols. Very carefully purified
2,4,6-tri- and pentachlorophenate were studied, in addition to a
commercial formulation of 2,3,4,6-tetra-chlorophenate. The analytical
method used in this study was not isomer specific, but the following
conclusions can be drawn concerning the formation of PCDDs by thermal
reactions:

(a) the expected dimerization products and the products formed
in the "Smiles rearrangement" are the major PCDDs;
(b) no other thermal isomerization of the PCDDs formed can be
observed;
(c) no formation of higher chlorinated PCDDs can be observed;
(d) octaCDD and other higher chlorinated PCDDs yield lower
chlorinated dioxins in a nonspecific dechlorination
reaction;
(e) a series of PCDFs was also observed.

It has been found that PCBs can be converted to PCDFs under
pyrolytic conditions. The pyrolysis of commercial PCBs in sealed
quartz ampoules in the presence of air yielded about 30 major, and
more than 30 minor, PCDFs. The optimal yield of PCDFs was about 10%,
calculated on the amount of PCB decomposed. Thus, uncontrolled burning
of PCBs can be an important environmental source of hazardous PCDFs.
Therefore, it was recommended (Buser et al., 1978a, 1978d) that all
destruction of PCB-contaminated waste using incinerators must be
carefully controlled. In the temperature range 300-400 °C, the
conversion yield seems to be in the part-per-million range (Morita et
al., 1978).

Buser & Rappe (1979) studied the pyrolysis of 15 individual
synthetic PCB congeners and showed that the formation of PCDFs can
follow several competing reaction pathways. In another study where a
series of chlorobenzenes were pyrolyzed in the same way, Buser (1979)
found that significant amounts (> 1%) of PCDDs and PCDFs were formed.
A complex mixture of isomers of PCDDs and PCDFs was found, suggesting
several reaction routes. Using the same technique as above, Lindahl et
al. (1980) studied the thermal decomposition of polychlorinated
diphenyl ethers. Both PCDDs and PCDFs were formed, involving several
pathways. The temperature range was 500-600 °C and the yields varied
from 0.1 to 4.5%.

Bergman et al. (1984) studied the thermal degradation of two
polychlorinated alkanes containing 59% and 70% chlorine, respectively,
and also a commercial chlorinated paraffin containing 70% chlorine.
Their studies indicated the presence of at least mono- and diCDFs.

Ahling et al. (1978) reported that chlorinated benzenes can be
found in the pyrolysis of PVC.

Direct evidence for the conversion of PVC to PCDDs and PCDFs has
recently been reported by Marklund et al. (1986). They found that
laboratory pyrolysis of PVC resulted in the formation of PCDDs and
PCDFs, mainly hexa- and heptaCDDs, and tetra- to heptaCDFs. In some
cases, the pattern of isomers seemed to be similar to those found in
municipal and hazardous waste incinerators, e.g. the pentaCDFs (Rappe
et al., 1987).

The data discussed in this section are summarized in Table 16.

3.5.2 Incineration of municipal waste

For some time, emissions from municipal incinerators, heating
facilities, and thermal power plants have been the subject of concern.
Whereas previously the emission of dust, smoke, toxic metals, and
noxious gases were of prime concern, the presence of potentially
hazardous organic compounds from these emissions has been recognized
only recently. Lahaniatis et al. (1977) reported the presence of
chlorinated organic compounds (chlorinated aliphatics, benzenes, PCBs,
and pesticides) in fly ash from a municipal incinerator.

Olie et al. (1977) reported the occurrence of PCDDs and PCDFs in
fly ash from three municipal incinerators in the Netherlands. Their
results indicated the presence of up to 17 PCDD peaks, but isomer
identification and quantification was not possible due to the lack of
synthetic standards. Buser & Bosshardt (1978) studied fly ash from a
municipal incinerator and an industrial heating facility, both in
Switzerland. In the former, the level of PCDDs was 0.2 µg/g and of
PCDFs 0.1 µg/g. In the industrial incinerator, the levels were 0.6
µg/g and 0.3 µg/g, respectively.

During the period 1978-1982 a series of papers, reports, and
reviews were published confirming the original findings of Olie et al.
(1977) and Buser & Bosshardt (1978) regarding fly ash. Less data have
been published on the levels of PCDDs and PCDFs in other incineration
by-products, e.g., particulates and flue gas condensate, and in total
flue gas, which are the true emissions (Marklund et al., 1986).

A risk evaluation should be based on the emission levels of PCDD
and PCDF isomers found in isomer-specific analyses using validated
sampling and clean-up methods. However, in many studies non-validated
sampling and analytical methods are used and the results are given in

terms of total levels of tetra-, penta-, hexa-, hepta-, and octaCDDs
and CDFs. The value of such studies is limited, particularly in this
situation where the number of isomers is quite large. More than 30
PCDDs and 60 PCDFs have been found in fly ash samples (Buser et al.,
1978b, 1978c).

In March 1986, a working group of experts convened by the World
Health Organization Regional Office for Europe reviewed the available
data on emissions of PCDDs and PCDFs from municipal solid-waste (MSW)
incinerators. It was found that the origin of these compounds was not
completely understood, but they appear to result from complex thermal
reactions occurring during periods of poor combustion. Because of
their high thermal stability, the PCDDs and PCDFs can be destroyed
only after adequate residence times at temperatures above 800 °C
(WHO/EURO, 1987).

Available data on total emissions of PCDDs and PCDFs from tests
on MSW incinerators range between a few and several thousand ng/Nm³
dry gas at 10% carbon dioxide (CO₂). The working group prepared a
table giving a range of estimated isomer specific emissions for those
isomers of major concern with respect to MSW incinerators operating
under various conditions (Table 17).

The emissions tabulated in column 1 are those which the working
group considered to be achievable in the most modern, highly
controlled, and carefully operated plants in use at the present time.
Such results do not represent what is considered to be achievable by
the use of acid gas cleaning equipment; use of such equipment should
result in much lower values (probably at least one order of
magnitude). The results given in column 1 are not representative of
emissions that might be expected from such plants during start-up or
during occasional abnormal conditions. Emission levels listed in
column 2 were considered by the working group to be indicative of the
higher limit of emissions from modern MSW incinerators. These plants
might experience such emissions during start-up or during occasional
upset conditions. Consequently, the majority of the available
concentration data falls between columns 1 and 2. Some of the data
reviewed has shown that the figures in column 2 should not be
considered an absolute maximum. However, most existing plants, if
carefully operated, will have PCDD and PCDF emisions in the range
between columns 1 and 2.

The highest values for MSW incinerators (column 3) were obtained
by multiplying the values in column 2 by a factor of 5. Column 3
includes emission data that were reported to the working group from
all tests and under all circumstances. Generally, these emission
levels are associated with irregular or unstable operating conditions,
high moisture content of the MSW, low combustion or afterburner
temperatures, less than adequate technologies, etc.

Table 16. Formation of PCDDs and PCDFs by thermal processes

Precursor Conditions Products

2,4,5-T salt Pyrolysis 2,3,7,8-tetraCDD
2,4,5-T (vegetation) Pyrolysis No TCDD
Burning No TCDD
Cl-phenate Burning PCDDs^a + PCDFs
PCBs Pyrolysis PCDFs^b
PCBz^c Pyrolysis PCDFs + PCDDs^d
Cl-Diphenyl ethers Pyrolysis PCDFs + PCDDs
Cl-Alkanes (Paraffins) Pyrolysis PCDFs
PVC Pyrolysis PCDDs + PCDFs

^a = PCDDs formed by dimerization and a non-specific dechlorination.
^b = other products: hexa- and pentaCBs.
^c = polychlorinated benzenes.
^d = other products: PCBs, polychlorinated naphthalenes.

The working group was aware of both lower and higher emission
levels than those included in Table 17. However, it was felt that the
values included in Table 17 were likely to be representative of
emissions from current facilities (WHO/EURO, 1987).

Of special importance is the observation that the emission of
1,2,3,7,8-pentaCDD normally exceeds the emission of 2,3,7,8-tetraCDD
by a factor of three to ten.

3.5.3 Incineration of sewage sludge

Sludge from municipal waste water treatment plants may be
incinerated after being dewatered. The WHO working group (see 3.5.2)
reviewed the available data from municipal sewage sludge (MSS)
incinerators, and found that PCDD and PCDF emissions from this type of
plant were generally lower than emissions from MSW incinerators (see
Table 17, column 4) (WHO, 1986).

3.5.4 Incineration of hospital waste

Doyle et al. (1985) claimed that the incomplete combustion of
certain hospital waste containing halogenated organics could produce
high levels of PCDDs and PCDFs. They found the mean values of total
PCDDs to be 69 ng/m³ and total PCDFs to be 156 ng/m³. No
isomer-specific data seems to be available. Hagenmaier et al. (1986)
reported the analyses of stack gas from 10 hospital waste incineration
plants. The mean value of 2,3,7,8-tetraCCD emitted was 0.28 ng/m³,
the mean of all TCDDs being 20 ng/m