Alpha- and beta-hexachlorocyclohexanes (EHC 123, 1991)

INTERNATIONAL PROGRAMME ON CHEMICAL SAFETY

ENVIRONMENTAL HEALTH CRITERIA 123

ALPHA- and BETA-HEXACHLOROCYCLOHEXANES

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

First draft prepared by Dr. G.J. van Esch,
Bilthoven, The Netherlands

Published under the joint sponsorship of the United Nations
Environment Programme, the International Labour Organisation, and the
World Health Organization

World Health Organization
Geneva, 1992

The International Programme on Chemical Safety (IPCS) is a joint
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of the biological action of chemicals.

WHO Library Cataloguing in Publication Data

Alpha- and Beta-hexachlorocyclohexanes.

(Environmental health criteria ; 123)

1.Benzene hexachloride - adverse effects 2.Benzene hexachloride -
toxicity 3.Environmental exposure 4.Environmental pollutants
I.Series

ISBN 92 4 157123 3 (NLM Classification: QV 633)
ISSN 0250-863X

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CONTENTS

ENVIRONMENTAL HEALTH CRITERIA FOR ALPHA- AND BETA-HEXACHLOROCYCLOHEXANES

A. ALPHA-HEXACHLOROCYCLOHEXANE

B. BETA-HEXACHLOROCYCLOHEXANE

CONCLUSIONS AND RECOMMENDATIONS FOR PROTECTION OF HUMAN HEALTH AND THE
ENVIRONMENT (ALPHA- AND BETA-HEXACHLOROCYCLOHEXANES)

FURTHER RESEARCH (ALPHA- AND BETA-HEXACHLOROCYCLOHEXANES)

PREVIOUS EVALUATIONS BY INTERNATIONAL BODIES

REFERENCES

APPENDIX 1. CHEMICAL STRUCTURE

RESUME ET EVALUATION

1. Alpha-hexachlorocyclohexane
2. Béta-hexachlorocyclohexane

CONCLUSIONS ET RECOMMANDATIONS

RECHERCHES A EFFECTUER (ALPHA- ET BETA-HEXACHLOROCYCLOHEXANES)

RESUMEN Y EVALUACION

1. Alpha-hexaclorociclohexano
2. Beta-hexaclorociclohexano

CONCLUSIONES Y RECOMENDACIONES

OTRAS INVESTIGACIONES (ALPHA- Y BETA-HEXACLOROCICLOHEXANOS)

WHO TASK GROUP MEETING ON ENVIRONMENTAL HEALTH CRITERIA FOR ALPHA-
AND BETA-HEXACHLOROCYCLOHEXANES

Members

Dr S. Dobson, Institute of Terrestrial Ecology, Monkswood Experimental
Station, Abbots Ripton, Huntingdon, United Kingdom

Dr M. Herbst, ASTA Pharma A.G., Frankfurt, Germany (Joint Rapporteur)

Professor J.S. Kagan, Department of General Toxicology and
Experimental Pathology, All-Union Scientific Research Institute of
Hygiene and Toxicology of Pesticides, Polymers, and Plastics, Kiev,
USSR (Vice-Chairman)

Dr S.G.A. Magwood, Pesticides Division, Environmental Health Centre,
Health & Welfare Canada, Tunney's Pasture, Ottawa, Ontario, Canada

Professor Wai-On Phoon, National Institute of Occupational Health and
Safety, University of Sydney, Sydney, Australia (Chairman)

Dr J.F. Risher, US Environmental Protection Agency, Environmental
Criteria and Assessment Office, Cincinnati, Ohio, USA

Dr Y. Saito, Division of Foods, National Institute of Hygienic
Sciences, Setagaya-ku, Tokyo, Japan

Dr V. Turusov, Laboratory of Carcinogenic Substances, All-Union Cancer
Research Centre, Moscow, USSR

Dr G.J. van Esch, Bilthoven, The Netherlands (Joint Rapporteur)

Representatives of Non-Governmental Organizations

Dr P.G. Pontal, International Group of National Associations of
Manufacturers of Agrochemical Products (GIFAP), Rhône-Poulenc Agro,
Lyon, France

Observers

Dr A.V. Bolotny, All-Union Scientific Research Institute of Hygiene
and Toxicology of Pesticides, Polymers, and Plastics, Kiev, USSR

Dr D. Demozay, International Centre for Study on Lindane (CIEL),
Rhône-Poulenc Agro, Lyon, France

Secretariat

Dr G.J. Burin, International Programme on Chemical Safety, World
Health Organization, Geneva, Switzerland

Dr K.W. Jager, International Programme on Chemical Safety, World
Health Organization, Geneva, Switzerland (Secretary)

Dr V.A. Rezepov, Centre for International Projects, USSR State
Committee for Environmental Protection, Moscow, USSR

NOTE TO READERS OF THE CRITERIA MONOGRAPHS

Every effort has been made to present information in the criteria
monographs as accurately as possible without unduly delaying their
publication. In the interest of all users of the environmental health
criteria monographs, 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.

* * *

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 or
7985850).

ENVIRONMENTAL HEALTH CRITERIA FOR ALPHA- AND BETA-HEXACHLOROCYCLOHEXANES

A WHO Task Group on Environmental Health Criteria for Alpha- and
Beta-hexachlorocyclohexanes met in Moscow from 20 to 24 November 1989.
The meeting was convened with the financial assistance of the United
Nations Environment Programme (UNEP) and was hosted by the Centre for
International Projects (CIP), USSR State Committee for Environmental
Protection. Dr V.A. Rezepov opened the meeting on behalf of the CIP
and welcomed the participants. Dr K.W. Jager welcomed the participants
on behalf of the three IPCS cooperating organizations (UNEP/ILO/WHO).
The Task Group reviewed and revised the draft criteria monograph and
made an evaluation of the risks for human health and the environment
from exposure to alpha- and beta-hexa-chlorocyclohexanes.

The first and second drafts of this monograph were prepared by
Dr G.J. van Esch (on behalf of the IPCS). Dr K.W. Jager and Dr P.G.
Jenkins, both members of the IPCS Central Unit, were responsible for
the overall scientific content and technical editing, respectively.

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

ABBREVIATIONS

cGMP cyclic guanosine monophosphate
CNS central nervous system
EEG electroencephalogram
EMG electromyogram
FDA Food and Drug Administration (USA)
FSH follicle-stimulating hormone
GABA gamma-aminobutyric acid
GGT gamma-glutamyltransferase
GLC gas-liquid chromatography
HCB hexachlorobenzene
HCCH hexachlorocyclohexene
HCH hexachlorocyclohexane
ip intraperitoneal
LH luteinizing hormone
MTD maximum tolerated dose
nd not detected
NOEL no-observed-effect level
PCB polychlorinated biphenyl
PCCH pentachlorocyclohexane
PIC picrotoxin
PTZ pentylenetetrazole
SEM smooth endoplasmic reticulum

PART A

ENVIRONMENTAL HEALTH CRITERIA FOR
ALPHA-HEXACHLOROCYCLOHEXANE

CONTENTS

ENVIRONMENTAL HEALTH CRITERIA FOR ALPHA-HEXACHLOROCYCLOHEXANE

1. SUMMARY AND EVALUATION

1.1. General properties
1.2. Environmental transport, distribution, and
transformation
1.3. Environmental levels and human exposure
1.4. Kinetics and metabolism
1.5. Effects on organisms in the environment
1.6. Effects on experimental animals and
in vitro test systems
1.7. Effects on humans

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS

2.1. Identity of primary constituent
2.2. Physical and chemical properties
2.3. Analytical methods

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION

4.1. Transport and distribution between media
4.2. Biotransformation
4.2.1. Biodegradation
4.2.2. Abiotic degradation
4.2.3. Bioaccumulation/biomagnification
4.2.3.1 Algae
4.2.3.2 Invertebrates
4.2.3.3 Fish
4.2.3.4 Bioconcentration in humans
4.3. Isomerization

5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

5.1. Environmental levels
5.1.1. Air
5.1.2. Water
5.1.2.1 Rain water
5.1.2.2 Fresh water
5.1.2.3 Sea water

5.1.3. Soil/sediment
5.1.3.1 Dumping grounds
5.1.4. Food and feed
5.1.5. Terrestrial and aquatic organisms
5.1.5.1 Plants
5.1.5.2 Fish and mussels
5.1.5.3 Birds
5.1.5.4 Mammals
5.2. General population exposure
5.2.1. Total-diet studies
5.2.2. Air
5.2.3. Concentrations in human samples
5.2.3.1 Blood
5.2.3.2 Adipose tissue
5.2.3.3 Breast milk

6. KINETICS AND METABOLISM

6.1. Absorption and elimination
6.2. Distribution
6.3. Metabolic transformation
6.3.1. Rat
6.3.2. Bird
6.3.3. Human
6.4. Retention and biological half-life

7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS

7.1. Single exposure
7.1.1. Acute toxicity
7.2. Short-term exposure
7.2.1. Oral
7.2.2. Other routes
7.2.2.1 Intravenous
7.2.2.2 Subcutaneous
7.3. Skin and eye irritation; sensitization
7.4. Long-term exposure
7.4.1. Rat oral study
7.5. Reproduction, embryotoxicity, and teratogenicity
7.6. Mutagenicity and related end-points
7.7. Carcinogenicity
7.7.1. Mouse
7.7.2. Rat
7.7.3. Initiation-promotion
7.7.4. Mode of action
7.8. Special studies
7.8.1. Effect on liver enzymes
7.8.2. Neurotoxicity

8. EFFECTS ON HUMANS

8.1. Acute toxicity - poisoning incidents
8.2. General population
8.3. Occupational exposure

9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD

9.1. Algae
9.2. Protozoa
9.3. Invertebrates
9.3.1. Acute toxicity
9.3.2. Short- and long-term toxicity
9.3.2.1 Crustaceae
9.3.2.2 Molluscs
9.4. Fish
9.4.1. Acute toxicity
9.4.2. Short- and long-term toxicity
9.5. Terrestrial organisms

1. SUMMARY AND EVALUATION

1.1 General properties

Alpha-hexachlorocyclohexane (alpha-HCH) is a major by-product
(65-70%) in the manufacture of lindane (> 99% gamma-HCH). Its
solubility in water is low, but it is very soluble in organic solvents
such as acetone, chloroform, and xylene. It is a solid with a low
vapour pressure. The n-octanol/water partition coefficient (log
P_ow) is 3.82. It is an environmental pollutant.

Alpha-HCH can be determined separately from the other isomers by
gas chromatography with electron capture detection and other methods
after extraction by liquid/liquid partition and purification by column
chromatography.

1.2 Environmental transport, distribution, and transformation

Biodegradation and abiotic degradation (dechlorination) by
ultraviolet irradiation occur in the environment and produce,
respectively, delta-3,4,5,6-tetrachloro-hexene and
pentachlorocyclohexene. This breakdown process is slower than in the
case of lindane. The persistence of alpha-HCH in soil is determined by
environmental factors such as the action of microorganisms, organic
matter content, and co-distillation and evaporation from soils. No
isomerization occurs from lindane to alpha-HCH.

Rapid bioconcentration takes place in microorganisms (the
bioconcentration factor equals 1500-2700 on a dry-weight basis, or
approximately 12 000 on a lipid basis within 30 min), invertebrates
(60-2750 (dry weight basis) or > 8000 (lipid basis) within 24-72 h),
and fish (313-1216 within 4-28 days; up to 50 000 in the River Elbe).
However, biotransformation and elimination is also fairly rapid in
these organisms (15 min to 72 h).

1.3 Environmental levels and human exposure

Alpha-HCH is found in air over the oceans at a concentration of
0.02-1.5 ng/m³. In Canada, it was found to be present in rain water
at a concentration of 1-40 ng/litre, but only traces were present in
snow.

During the period 1969-1974, the River Rhine and its tributaries
contained alpha-HCH levels of 0.01-2.7 µg per litre, but more recently
the levels have been below 0.1 µg/litre. In the River Elbe, levels
decreased from a mean of 0.023 µg/litre in 1981 to below 0.012 µg per
litre in 1988. Selected rivers in the United Kingdom were found in
1966 to contain 0.001-0.43 µg/litre. Alpha-HCH has been found in North
Frisian Wadden Sea sediment at concentrations of between 0.3 and
1.4 µg/kg (0.002 µg per litre in water).

Alpha-HCH levels in different plant species from various
countries varied from 0.5-2140 µg/kg on a dry-weight basis, but were
much higher in polluted areas. Even in Antarctica, levels ranging from
0.2-1.15 µg/kg have been found.

Alpha-HCH is regularly detected in fish and aquatic
invertebrates, as well as in ducks, herons, and barn-owls. In
reindeer and Idaho moose, living in areas with negligible use of
pesticides, average amounts of alpha-HCH of approximately 70-80 µg/kg
were found in the subcutaneous fat. The adipose tissue of Canadian
polar bears contained 0.3-0.87 mg alpha-HCH/kg (on a fat basis).

In a number of countries, important food items have been analysed
for the presence of alpha-HCH. The levels, mainly in fat-containing
food products, ranged up to 0.05 mg/kg product, except in milk and
milk products (up to 0.22 mg/kg) and in fish and processed meat
products (up to 0.5 mg/kg on a fat basis). A slow decrease over the
years has been noted.

Food is the main source for general population exposure to
alpha-HCH. In total-diet studies in the Netherlands and the United
Kingdom, mean concentrations of 0.01 and 0.002-0.003 mg/kg food,
respectively, were found. The United Kingdom data indicate a downward
trend since 1967. In the USA, the average daily intake of alpha-HCH
was 0.009-0.025 µg/kg body weight during the period 1977-1979, and
0.003-0.016 µg/kg body weight during the period 1982-1984.

In a few countries, the concentration of alpha-HCH has been
determined in human blood, serum, or plasma. The mean (in some cases
median) concentration was < 0.1 µg per litre (ranging from
undetectable levels to 0.6 µg per litre). In one country, however, a
mean concentration of 3.5 (range 0.1-15.0) µg/litre was reported.
Alpha-HCH was detected in approximately one third of the blood
samples.

The concentrations in human adipose tissue and breast milk are
reported to be low (respectively < 0.01-0.1 and < 0.001-0.04 mg/kg
on a fat basis). Total-diet studies have shown daily intake levels
of the order of 0.01 µg/kg body weight per day or lower. These
concentrations are decreasing slowly over the years.

Alpha-HCH appears to be a universal environmental contaminant.
Concentrations are only decreasing slowly, in spite of measures taken
to prevent its spread into the environment.

1.4 Kinetics and metabolism

In rats, alpha-HCH is rapidly and almost completely absorbed from
the gastrointestinal tract. After intraperitoneal injection,
approximately 40-80% of the alpha-HCH was excreted via the urine and
5-20% via the faeces. In rats, the highest concentrations have been

found in liver, kidneys, body fat, brain and muscles, and substantial
deposition occurs in fatty tissue. The alpha-HCH concentrations in the
liver of sucklings were twice as high as those observed in the liver
of the mothers. In rats, the brain to blood and depot fat to blood
ratios were 120:1 and 397:1, respectively.

The biotransformation of alpha-HCH in rats involves
dechlorination. The major urinary metabolite is 2,4,6-tri-
chlorophenol; other identified metabolites include 1,2,4-, 2,3,4-, and
2,4,5-trichlorophenol and 2,3,4,5- and 2,3,4,6-tetrachlorophenol.
1,3,4,5,6-Pentachlorocyclohex-1-ene has been found in rat kidneys and
also in in vitro studies on chicken liver. A glutathione conjugate
is formed in the liver.

The half-life for clearance from the fat depot is 6.9 days in
female rats and 1.6 days in males.

1.5 Effects on organisms in the environment

Alpha-HCH has low toxicity for algae, 2 mg/litre generally being
the no-observed-effect level.

In a long-term study, Daphnia magna showed a no-observed-effect
level of 0.05 mg/litre. Alpha-HCH is moderately toxic for
invertebrates and fish. The acute L(E)C₅₀ values for these
organisms are in the order of 1 mg/litre. In short-term studies with
guppies and Oryzia latipes, 0.8 mg/litre was without effect.

In three-month studies with Salmo gairdneriat dose levels of
10-1250 mg/kg diet, there were no effects on mortality, behaviour,
growth, or enzyme activities in liver and brain.

Short- and long-term studies with a snail (Lymnea stagnalis)
showed an EC₅₀ (based on mortality and immobilization) of
1200 µg/litre. Inhibition of egg production occurred at a
concentration of 250 µg/litre. A 50% reduction in the overall
reproductivity was found at 65 µg/litre.

No data are available on effects on populations and ecosystems.

1.6 Effects on experimental animals and in vitro test systems

The acute oral LD₅₀ values for mice lie between 1000-4000 and
for rats between 500-4670 mg/kg body weight. The poisoning signs are
mainly those of stimulation of the central nervous system.

A 90-day study with rats showed growth depression at a
concentration of 250 mg/kg diet. Histological and enzyme level
changes in the liver indicated enzyme induction at 50 mg/kg or more.
At these dose levels there were also indications of immunosuppression.

Liver weights were already increased at 10 mg/kg diet (equivalent to
0.5 mg/kg body weight). The no-observed-adverse-effect level in this
study appeared to be 2 mg/kg diet (equivalent to 0.1 mg/kg body weight
per day).

No adequate long-term toxicity studies or reproduction and
teratogenicity studies have been reported.

Studies with various strains of Salmonella typhimurium yielded
no evidence of mutagenicity either with or without metabolic
activation. Tests with Saccharomyces cerevisiae were also negative,
but a test for unscheduled DNA synthesis in rat hepatocytes in vitro
gave an equivocal result.

Studies to determine carcinogenic potential have been carried out
with mice and rats at dose levels from 100 to 600 mg/kg diet.
Hyperplastic nodules and/or hepatocellular adenomas were found in
studies on mice. In one study the dose levels exceeded the maximum
tolerated dose. Two mice studies and one rat study, using dose levels
of up to 160 mg/kg diet in mice and 640 mg/kg diet in rats, did not
show any increase in the incidence of tumours.

The results of the studies on initiation-promotion and mode of
action and the mutagenicity studies indicate that the
alpha-HCH-induced tumorigenicity observed in mice has a non-genetic
mechanism.

Alpha-HCH has been shown to cause a clear increase in the
activity of liver enzymes even at 5 mg/kg diet (equivalent to
0.25 mg/kg body weight). A dose of 2 mg/kg body weight did not affect
aminopyrine demethylation or the DNA content of the liver.

1.7 Effects on humans

When workers at a lindane-producing factory, with a geometric
mean exposure of 7.2 years (1-30), were investigated, it was concluded
that occupational HCH exposure did not induce signs of neurological
impairment or perturbation of "neuromuscular function".

2. IDENTITY, PHYSICAL AND CHEMICAL PROPERTIES, ANALYTICAL METHODS

2.1 Identity of primary constituent

Common name Alpha-hexachlorocyclohexane (alpha-HCH)

Chemical formula C₆H₆Cl₆

Chemical Alpha-HCH is a stereoisomer of gamma-
structure HCH, the active ingredient of lindane
(see Appendix 1) (> 99% gamma-HCH). It differs in the
spatial orientation of the hydrogen and
chlorine atoms on the carbon atoms:

Relative
molecular mass 290.9

CAS chemical 1alpha,2alpha,3ß,4alpha,5ß,6ß-hexachloro-
name cyclohexane

Common
synonyms Alpha-benzenehexachloride (alpha-BHC)

CAS registry
number 319-84-6

RTECS registry
number GV3500000

2.2 Physical and chemical properties

Some physical and chemical properties are summarized in Table 1.

Table 1. Some physical and chemical properties of alpha-
hexachlorocyclohexane

Melting point 158°C

Boiling point 288°C

Vapour pressure (20°C) 2.67 Pa (0.02 mmHg)

Relative density (20°C) 1.87 g/cm³

Solubility
water (28°C) 2 mg/litre
organic solvents (20°C) acetone 139 g/litre
chloroform 63 g/litre
ethanol 18 g/litre
petroleum ether 7-13 g/litre
xylene 85 g/litre

Stability considerable stability in acids,
unstable in alkaline conditions

n-Octanol/water partition
coefficient (log P_ow) 3.82

2.3 Analytical methods

Hildebrandt et al. (1986) and Wittlinger & Ballschmiter (1987)
described in detail the appropriate analytical methods, i.e. air
sampling by adsorption, extraction, purification, and determination
using high resolution gas chromatography. Sampling was conducted by
pumping air first through a glass fiber filter and then a layer of
silica gel. An internal standard was used. The extraction was
carried out with dichloromethane, and the extract was evaporated.
Preseparation was on silica gel and elution with a mixture of hexane
and dichloromethane. For the determination, use was made of high
resolution capillary gas chromatography with electron capture
detection and a mass selective detector.

Eder et al. (1987) described in detail three different analytical
methods for the determination of HCHs in sediments. Sediments are
extracted with a solvent or mixture of solvents and are concentrated
or fractionated. The alpha-HCH is determined by gas chromatography
with electron capture detection or other methods.

3. SOURCES OF HUMAN AND ENVIRONMENTAL EXPOSURE

Alpha-HCH does not occur naturally. It is released to the
environment as a result of the use of technical-grade HCH and the
inappropriate disposal of the residue resulting from the purification
of lindane.

Alpha-HCH is basically a by-product (and impurity) in the
manufacturing of lindane (> 99% gamma-HCH). Technical-grade HCH,
which is synthesized from benzene and chlorine in the presence of
ultraviolet light, consists of:

65-70% alpha-HCH
7-10% beta-HCH
14-15% gamma-HCH (lindane)
approx. 7% delta-HCH
approx. 1-2% epsilon-HCH
approx. 1-2% other components

Purification of lindane produces a residue, consisting almost
entirely of non-insecticidal HCH isomers (mainly alpha- and beta-),
which can be used as an intermediate for the production of
trichlorobenzene and other chemicals.

Alpha- and beta-HCH have been used in mixtures with gamma-HCH (as
"HCH" or "fortified HCH") in agriculture and in wood protection.

4. ENVIRONMENTAL TRANSPORT, DISTRIBUTION, AND TRANSFORMATION

4.1 Transport and distribution between media

MacRae et al. (1967) studied the persistence and
bio-degradability of alpha-HCH in two clay soils. The rate of
treatment was 15 mg/kg soil, and incubation periods of 0, 15, 30, 50,
70, and 90 days were used. Only very small amounts of alpha-HCH could
be detected in non-sterilized soils after 70 days, indicating a low
level of persistence and biodegradation. However, the losses were
much slower in sterilized soils, and were probably due to
volatilization.

Tsukano (1973) studied the factors affecting the disappearance of
alpha-HCH from rice field soil after granular application
(0.05 mg/litre) to the surface water. The surface water and soil were
analysed at intervals, and alpha-HCH was found to disappear rapidly
with a half-life of about 5 days. Following translocation of
alpha-HCH (1 mg/litre) onto flooded levelled soil, a decrease in the
level in water and steady increase in the level in soil occurred.
After 7 days the concentration in soil reached a maximum. Data from a
soil column study showed that alpha-HCH moved downwards with the
percolating water.

Suzuki et al. (1975) studied the persistence of alpha-HCH in
three different types of soil. The persistence was found to be
determined by environmental factors such as the action of
microorganisms, co-distillation, evaporation from soil, and the
contents of water and organic matter in the soil.

In a study by Wahid & Sethunathan (1979), the sorption and
desorption of alpha-HCH by 12 soils from rice-growing areas in India
were studied using ¹⁴C label. The soils showed striking differences
in their ability to adsorb alpha-HCH, the sorption values ranging from
40 to 95% of total added alpha-HCH. After oxidation of the soil with
hydrogen peroxide, the sorption was lower (5-46%). Organic matter was
the most important factor governing the sorption and desorption, but
pH, exchange acidity, exchangeable sodium and magnesium, and
electrical conductivity also affected the results.

Korte (1980) summarized the behaviour of alpha-HCH in the
environment, especially in soil and plants.

4.2 Biotransformation

4.2.1 Biodegradation

Heritage & MacRae (1977, 1979) investigated the degradation of
alpha-HCH (final concentration 5 mg/litre) by a washed suspension of
Clostridium sphenoidesin the absence of oxygen at 30°C. The
alpha-isomer was no longer detectable after 4 h. Apparently the

degradation proceeded via delta-3,4,5,6-tetrachlorocyclohexene
(delta-TCCH). Aerobically grown facultative anaerobes actively
dechlorinated ³⁶Cl-alpha-HCH during anaerobic incubation with
glucose, pyruvate or formate as substrates, but this dechlorination
was slower than in the case of lindane.

When incubation studies were performed under anaerobic or aerobic
conditions, the dechlorination of ³⁶Cl-labelled alpha-HCH by mixed
soil flora and by pure cultures of Citrobacter freundii, C.
butyricum, and C. pasteurianumwas 6.5%, 13.9%, 97.4%, and 53.2%,
respectively, within 6 days of incubation. Again, alpha-HCH degraded
more slowly than lindane (Jagnow et al., 1977).

Screening experiments to study the possible isomerization of
lindane to alpha-HCH, using C. freundii, Serratia marcescens,
Pseudomonas putida, and other bacterial species, gave negative
results (Haider, 1979).

Doelman et al. (1985) carried out laboratory studies on the
degradation of alpha-HCH, at a concentration of approximately
5300 mg/kg, in a polluted Dutch sandy loam soil with 6.5% organic
matter. They found during 20 weeks constant degradation rates of
10 mg/kg per day under anaerobic conditions and 14 mg/kg per day under
aerobic conditions. At a lower concentration (approximately
3900 mg/kg) the average degradation rate appeared to be higher
(24 mg/kg per day) under both aerobic and anaerobic conditions. The
degradation was ascribed to microbial processes.

Studies in 1986 on HCH-polluted soil (personal communication by
P. Doelman and A. Zehnder to the IPCS) indicate that alpha-HCH
degrades considerably better in aerobic conditions (aerated slurry)
than in anaerobic conditions (non-aerated slurry) both in the
laboratory and in soil in greenhouses (Slooff & Matthijsen, 1988).
Assuming the degradation process to be a first-order reaction, MacRae
et al. (1984) calculated from laboratory studies (soil with 4.0%
organic carbon) half-lives of 125 and 48 days under aerobic and
anaerobic conditions, respectively.

In a study by Doelman et al. (1988a), microbial soil sanitation
was applied to calcareous alkaline sandy loam soil that was polluted
with a mixture of HCH isomers. Under anaerobic conditions, microbial
degradation in the Dutch climate (soil temperature of 5-17°C) did not
occur, and even the low concentration of the easily degradable
gamma-HCH did not decrease.

Microbial soil sanitation of alpha-HCH-polluted calcareous sandy
loam soil systems has been investigated. The soil systems involved
were aerated moist soil and continuously aerated and intermittently
aerated soil slurries. Degradation of alpha-HCH appeared to proceed
according to a first-order reaction. It was fastest during the first
4 weeks, even though soil temperatures were lowest during this period.

The percentage degradation during the first 4 weeks was 40, 80, and
37%, respectively, for the three soil systems. The degradation rate
gradually decreased with time even if the temperature increased.
Addition of microbial biomass did not significantly affect the
alpha-HCH degradation. In a continuously aerated thick slurry system,
the alpha-HCH concentration was reduced from approximately 420 to
15 mg/kg. Thus, alpha-HCH degradation will occur in regions with a
temperate climate, provided that the soil is aerobic (Doelman et al.,
1988b).

A field investigation into the distribution of HCHs was carried
out by Chessells et al. (1988) using soil from an agricultural area
treated with BHC-20 (HCH composition: 70% alpha-HCH, 6.5% beta-HCH,
13.5% gamma-HCH, and 5% delta-HCH. Although the concentration of
alpha-HCH was the highest of the HCHs, the alpha-isomer disappeared
more rapidly than beta-HCH. Furthermore, soil organic carbon content
was found to be of primary importance. A significant decrease in
isomer concentration was observed when soil moisture content was high
and was attributed to microbial degradation favoured by these
conditions.

4.2.2 Abiotic degradation

Alpha-HCH is broken down by ultraviolet light but at a slower
rate than lindane. Ultraviolet irradiation, using a 15-watt low
pressure mercury lamp, of alpha-HCH in 2-propanol solution for 10 h
resulted in the production of an isomer of pentachlorocyclohexene.
This substance may be produced by hydrogen abstraction of the
radiation-induced pentachlorocyclohexyl radicals (Hamada et al.,
1982).

4.2.3 Bioaccumulation/Biomagnification

4.2.3.1 Algae

A study was carried out to determine the bioconcentration of
alpha-HCH by an alga (Cladophora) during a period of 48 h. At
concentrations of alpha-HCH in water of 4.4 and 31 µg/litre, the
bioconcentration factors were 341 and 180, respectively (Bauer, 1972).

In a study by Canton et al. (1975), Chlorella pyrenoidosacells
taken from a log-phase culture were exposed for 96 h to alpha-HCH
(> 95%) concentrations of 10, 50 or 800 µg/litre, and after 15, 30, and
180 min the cells were analysed. At all dosage levels the average
bioconcentration from water was about 200-fold (153-267). There
seemed to be a tendency for alpha-HCH to accumulate in the cytoplasm
rather than the cell wall. When the cells were subsequently placed in
clean water, the elimination was rapid (15 min).

When Canton et al. (1977) investigated the accumulation and
elimination of alpha-HCH (> 95%) in marine algae (Chlamydomonas and

Dunaliella) in studies lasting a few days, both processes were found
to take place rapidly, (i.e. in less than 30 min). The average
concentration factor was 2700 in Chlamydomonas and 1500 in
Dunaliella (on a dry weight basis) and was 12 000 and 13 000,
respectively, on a lipid basis. The accumulated alpha-HCH was found
primarily in the lipophyllic parts of the cells.

4.2.3.2 Invertebrates

In a study by Canton et al. (1978), Artemia was exposed to
alpha-HCH (> 95%) levels of 0.01, 0.05 or 0.25 mg/litre and sampled
after 0.5, 3, 24, 48, 72, and 96 h. Once equilibrium was reached, the
animals were transferred to alpha-HCH-free water and were sampled
after 0, 3, 24, 48, 96, and 144 h. The bioconcentration factor was
about 60-90 (8000-11 000 on a lipid basis), and equilibrium was
reached within 24 h. The elimination half-life was 48-72 h.

Ernst (1979) measured alpha-HCH bioconcentration factors in two
marine invertebrates, the mussel (Mytilus edulis) and the polychaete
(Lanice conchilega), of 105 and 2750, respectively, at 10°C and an
alpha-HCH concentration of 2-5 µg/litre. Species differences and the
lipid content of the animals appeared clearly to affect the
bioconcentration factor, whereas the effect of temperature seemed to
be minimal.

In a study by Yamato et al. (1983), the short-necked clam
(Venerupis japonica) rapidly absorbed alpha-HCH and the
concentration reached a plateau on the third day. The
bioconcentration factor was 161 at an alpha-HCH concentration of
1 µg/litre water. The alpha-HCH concentrations on day 6 in organs and
tissues were 0.060 and 0.029 mg/kg, respectively. After a 3-day
elimination period, the levels were 0.033 and 0.024 mg/kg,
respectively.

Mouvet et al. (1985) investigated the presence of alpha-HCH in
the aquatic moss Cinclidotus danubicus to examine the potential use
of this species as an indicator of chlorinated pollutants in fresh
water. The moss was sampled 0, 13, 24, and 51 days after having been
transplanted in a polluted river, and levels of 0.20-1.33 µg per litre
water were found 4 km downstream of an area of industrial discharge.
The levels of alpha-HCH in the moss were < 0.025, 0.04-0.57,
0.08-2.37, and 0.81 mg/kg dry weight, respectively, at the time
intervals indicated above.

4.2.3.3 Fish

Canton et al. (1975) studied the accumulation and elimination of
alpha-HCH by Chlorella, Daphnia, and Poecilia reticulata, and in
Chlorella-Daphnia and Daphnia-Poecilia reticulata systems. In this
food-chain study, the following concentration ratios were measured:

The direct uptake of alpha-HCH from contaminated water appeared to be
much greater than the uptake from contaminated food.

In a study with Salmo gairdneri, pellets containing alpha-HCH
(> 95%) levels of 0, 10, 50, 250, or 1250 mg/kg were fed to the fish,
and organs and tissues were analysed after 2, 4, 8, and 12 weeks.
There was a dose-related increase in the concentration of alpha-HCH
in the organs and tissues. After about 4-8 weeks (depending on the
type of tissue and dose level) a maximum concentration was reached,
which then slowly decreased. This suggests that after a few weeks a
balance is reached between the accumulation process (absorption of
alpha-HCH by the intestinal wall) and the elimination process (via the
gills and faeces). There is probably also a dilution effect resulting
from growth and biotransformation (Canton et al., 1975).

Ernst (1977) concluded from kinetic studies that biomagnification
of alpha-HCH does not occur. Compared with bioaccumulation from water
alone, the entry of alpha-HCH into the food chain Chlorella ->
Daphnia -> Poecilia (guppy) caused only a slight increase in
biomagnification in daphnids (factor 1.5), although in the case of the
guppies a greater increase in concentration ratio (3-4) was noted.

In a study by Canton et al. (1978), guppies (3-4 weeks old) were
exposed to alpha-HCH (> 95%) concentrations of 0.01, 0.05, or
0.14 mg/litre. When after 0.5, 3, 24, 48, 72, 96, and 120 h the
animals were analysed, the average concentration factor was about 500
for all alpha-HCH concentrations (about 17 000 on a lipid basis).
Equilibrium was reached within 24 h for the lower concentrations and
within 48 h at the highest concentrations. The elimination was rapid,
the initial concentration being halved in 10 h.

Sugiura et al. (1979) studied bioaccumulation in the carp
(Cyprinus carpio), brown trout (Salmo trutta fario), golden orfe
(Leuciscus idus melanotus), and guppy (Poecilia reticulata).
Alpha-HCH was dissolved in water to a concentration of 1 mg/litre
under steady-state conditions (time period not specified), and the
equilibrium bioconcentration factors for the four types of fish were
330, 605, 1216, and 588, respectively.

Based on the data given in section 5.1.5.2 concerning the
concentration of alpha-HCH in the muscle and fat of bream collected in
the River Elbe, the bioconcentration factor is between 10 000 and
50 000 (Arbeitsgemeinschaft für die Reinhaltung der Elbe, 1982).

In a study by Yamato et al. (1983), guppies (Poecilia
reticulata) rapidly bioaccumulated HCH isomers and the tissue
concentration reached a plateau on the fourth day (the alpha-HCH
concentration in the water was 1 µg per litre). The bioconcentration
factor (concentration in fish/concentration in water) was 706. The
concentration in the guppies decreased on the first day after the fish
were transferred to HCH-free water.

4.2.3.4 Bioconcentration in humans

Geyer et al. (1986) found that in industrialized countries more
than 90% of the exposure to HCHs derives from food. The mean
concentration of alpha-HCH in human adipose tissue (on a fat basis)
was found to be 0.03 mg/kg in the Federal Republic of Germany and
0.02 mg/kg in the Netherlands. The mean bioconcentration factor (on a
lipid basis), calculated on the basis of the concentration in the diet
(1.3 and 0.3 µg/kg, respectively) and levels in adipose tissue, was
20.0 ± 8 (range 11.5-32.5).

4.3 Isomerization

Deo et al. (1981) studied the isomerization of alpha-HCH in
sterile aqueous solution over a period of 4 weeks and found a slow
conversion of alpha-HCH to other HCH isomers.

5. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

5.1 Environmental levels

5.1.1 Air

Tanabe et al. (1982) found alpha-HCH in 24 samples of air over
the Western Pacific, Eastern Indian, and Antarctic Oceans at an
average concentration of 0.29 ng/m³ (0.022-1.4 ng/m³).

In a study by Strachan et al. (1980), samples of atmospheric
precipitation in the form of snow (1976; 17 samples) and rain (1976
and 1977; 81 samples) collected around the Canadian side of the Great
Lakes, as well as inland, were analysed. Alpha-HCH was found in the
snow samples as a trace (1 ng/litre) and in the rain samples at levels
of 1-40 ng/litre.

Air samples were taken near a road with heavy traffic, as well as
in a suburban residential area, near Ulm, in Germany. The alpha-HCH
levels were 0.22-1.3 ng/m³ in the location with heavy traffic and
0.11-1.1 ng/m³ in the rural area. It was concluded that the
concentrations in the lower troposphere under various meteorological
conditions reflect regional input and long-range transport (Wittlinger
& Ballschmiter, 1987).

In 1972, alpha-HCH air concentrations of 0.28 ng per m³ in
non-polluted areas of Germany, and 2.15 ng/m³ in the polluted Ruhr
area were determined (Hildebrandt et al., 1986).

The average concentration of alpha-HCH in 55 air samples
collected in Delft, the Netherlands, in 1979-1980 was 0.25 ng/m³
(maximum concentration: 1.2 ng/m³) (Slooff & Matthijsen, 1988).

5.1.2 Water

5.1.2.1 Rain water

Rain water sampled in 1983 in Bilt, the Netherlands, contained an
average alpha-HCH concentration of 0.01 (< 0.01-0.02) µg/litre
(Slooff & Matthijsen, 1988).

5.1.2.2 Fresh water

During the period 1969-1977, 1826 water samples were taken at 99
sampling sites in the Netherlands. The highest concentrations of
alpha-HCH were found in the River Rhine and its tributaries. The
concentrations varied between 0.01-0.3 µg/litre during the period
1969-1974, but in 1974 there was a sudden decrease and the subsequent
concentrations were all below 0.1 µg/litre. A sampling trip by boat
made along the River Rhine from Rheinfelden in Switzerland to
Rotterdam in the Netherlands proved that the source of alpha-, beta-,

and gamma-HCH was located in the upper Rhine. In the River Meuse, the
levels were all below 0.1 µg/litre during the period 1969-1977 (Wegman
& Greve, 1980).

Since 1969, alpha-, beta-, and gamma-HCH concentrations have been
measured regularly in the Rivers Rhine, Meuse, and West-Scheldt and in
other surface waters in the Netherlands. Alpha-HCH levels have been
below 0.05 µg per litre in the River Rhine since 1974/1975, and were
of the order of 0.02 µg/litre or less in the West-Scheldt during the
period 1973-1985. In the River Meuse, the concentration of alpha-HCH
was between 0.01-0.02 µg/litre. In other areas, for instance
agricultural and greenhouse horticulture areas, the levels of the
individual HCHs ranged from 0.01-1.0 µg/litre with incidental higher
peaks (up to 0.5 µg/litre) probably resulting from the use of lindane
(Slooff & Matthijsen, 1988).

Concentrations of HCH isomers in solution and in suspension
(particle-bound) in the Meuse and Rhine estuary were determined in
1974. The average concentrations of dissolved and suspended alpha-HCH
were 20 and 0-6 ng per litre, respectively. In 1981, the
concentration of dissolved alpha-HCH in coastal waters of the
Netherlands was 0.9-1.6 ng/litre, whereas that of suspended alpha-HCH
(only one measurement) was 5.3 ng/litre (Slooff & Matthijsen, 1988).

In 1970-1971, the levels of alpha-HCH were 0.66-1.5 µg/litre in
the surface water of the River Elbe near Hamburg, Germany, and
0.155-2.4 µg/litre in the River Rhine near Karlsruhe. However, a
significant decrease was observed in the mid-1970s. In 1974, 2.7 µg
per litre was found in the upper Rhine, but by 1976-1977 the levels
had decreased to 1-9 ng/litre (Hildebrandt et al., 1986).

The Arbeitsgemeinschaft der Elbe (the Elbe Study Group)
investigated the presence of alpha-HCH in the River Elbe from
Schnackenburg to the North Sea in 1981-1982 and found a mean
concentration of 0.023 (< 0.001-0.15) µg per litre. During the period
February to November 1988, the alpha-HCH concentration was
0.001-0.022 µg/litre (Arbeitsgemeinschaft der Elbe, 1988).

When certain rivers in Yorkshire, England, were analysed for
alpha-HCH in 1966, the concentration varied from 0.001 to
0.43 µg/litre. In 1968, the highest value was 0.543 µg/litre, and
the water from six other rivers contained an average of
0.001-0.004 µg/litre (highest level: 0.34 µg/litre) (Lowden et al.,
1969).

In Japan, 60 water samples were examined in 1974 and 0.1 µg
alpha-HCH/litre was detected in three of the samples (personal
communications by A. Hamada and by T. Onishi to the IPCS, July 1989).

5.1.2.3 Sea water

Atlas & Gias (1981); Bidleman & Leonard (1982); Oehme & Stray
(1982); and Oehme & Mano (1984) analysed sea water from areas such as
the North Pacific, Arabic Sea, Persian Gulf, Red Sea, Lillestrum, Bear
Island, and Spitsbergen. The alpha-HCH concentrations varied from
0.03 to 1.8 ngper litre (Slooff & Matthijsen, 1988).

In June-July 1986, the alpha-HCH in the surface water (5 m) of
the North Sea ranged from 1-2 ng/litre (Umweltbundesamt, 1989).

5.1.3 Soil/Sediment

Herrmann et al. (1984) studied the presence of alpha-HCH in
sediment along the Husum estuary and in the adjacent North Frisian
Wadden Sea. The mean concentrations varied in the different sampling
stations from 0.33 to 1.40 µg/kg sediment, while the concentrations in
bladder wrack(Fucus vesiculosus)varied from 0.7-1.2 µg/kg.

Edelman (1984) analysed 96 samples of the upper 10 cm of the soil
from 38 natural reserves in the Netherlands for alpha-HCH and
gamma-HCH. In 94 of the samples alpha-HCH was detected at levels
below 1 µg/kg (Slooff & Matthijsen, 1988).

When sediment from eight different rivers, harbours, and sites
close to dumping areas in the Netherlands were analysed for the
presence of alpha-, beta-, and gamma-HCH, the median alpha-HCH levels
were between 4 and 213 µg per kg dry matter (Slooff & Matthijsen,
1988).

In 1974, 60 sediment samples were analysed in Japan and 10 µg
alpha-HCH/kg was detected in five of the samples (personal
communications by A. Hamada and by T. Onishi to the IPCS, July 1989).

5.1.3.1 Dumping grounds

In the Netherlands, soil has been polluted with HCHs at various
locations as a result of their manufacture during the 1950s (spillage
during production, storage, and handling), and concentrations up to a
few grams of HCHs/kg dry soil have been found. Further pollution has
been caused by the dumping of chemical waste and its use in the
levelling of certain areas. From these dumping areas dispersal of the
chemical waste can occur by leaching or wind erosion from open storage
depots. In certain polluted areas, high concentrations of HCHs, mainly
alpha- and beta-HCH, have been found more than 2 m below ground level.
In 18 locations in the Netherlands, the average concentration of
alpha-HCH in sewage sludge in 1981 was between 5 and 70 µg/kg dry
matter. Pollution of ground water was also detected, but this was
restricted to the vicinity of the production areas. Horizontal
transportation of HCHs in ground water appeared to be limited (Slooff
& Matthijsen, 1988).

5.1.4 Food and feed

The presence of alpha-HCH in a number of important food items has
been determined in France by Laugel (1981). In milk and milk products
(2688 samples) the average level was 0.05 mg/kg (ranging from
undetectable to 0.22 mg/kg), in meat (37 samples) it was 0.01 mg/kg
(ranging from undetectable to 0.02 mg/kg), and in animal fat (67
samples) it was 0.02 mg/kg product (ranging from undetectable to
0.06 mg/kg. In other food items alpha-HCH was not detectable
(< 0.005 mg/kg).

Table 2 gives the mean alpha-HCH levels in a large number of
samples of various food items from the Federal Republic of Germany
reported by Hildebrandt et al. (1986).

Table 2. Alpha-hexachlorocyclohexane concentrations (mg/kg)
in various food items^a

Food items 1973-78 1979-83 1973-83

Meat^b 0.003-0.02

Meat products^b 0.007-0.037
(0.26)^e

Animal fat^b 0.003-0.008
(0.09)^e

Game^b 0.019-0.367

Poultry^b 0.003-0.004 0.003-0.016
(0.17)^e

Chicken eggs < 0.001-0.003

Fish 0.002-0.011

Milk and milk
products^b 0.015^e 0.01-0.03

Cow's milk^b,c 0.004

Butter^b,d 0.02-0.03

Table 2 (contd)

Food items 1973-78 1979-83 1973-83

Vegetable oil and
margarine^b 0.01

Oil seeds, nuts,
pulses 0.001-0.042

Fruit, vegetables, < 0.0001
potatoes

Cereals 0.0002-0.007

Cereal products up to 0.14

^a From: Hildebrandt et al. (1986).
^b Determinations made on a fat basis
^c WHO (1986).
^d Anon (1984).
^e Maximum value

In six samples of cows milk collected from six locations in
Switzerland, the levels of alpha-HCH were 9.5-27 mg/kg on a fat basis
(Rappe et al., 1987).

Skaftason & Johannesson (1979) analysed 35 samples of butter from
Iceland during 1968-1970 and found a level of mean alpha-HCH of 87 ±
38 µg/kg. In 1974-1978, 32 samples were studied and all contained
alpha-HCH, the mean concentration being 58 ± 21 µg/kg.

In a total-diet study in the United Kingdom, 24 samples of each
food group were analysed for alpha-HCH. The following concentrations
(mean and range) were found: bread, not detected (nd); other cereal
products, < 0.0005 (nd-0.002); carcass meat, < 0.0005 (nd-0.006);
offal, < 0.0005 (nd-0.007); meat products, eggs, green vegetables,
potatoes, fresh fruit, nd; poultry, 0.003 (nd-0.025); fish, 0.0005
(nd-0.008); oil and fats, 0.0005 (nd-0.003); milk, 0.0005 (nd-0.002);
dairy products, 0.006 (nd-0.02) mg/kg product. Imported meat products
were also analysed during the period 1981-1983, and concentrations of
up to 0.5 mg/kg were measured. Imported retail cereal products
collected in 1982 contained alpha-HCH levels of up to 0.03 mg/kg and
animal feed stuffs collected in 1984 had levels of up to 0.02 mg/kg
(HMSO, 1986).

Various types of pulses were analysed during the period
1986-1987, and 31 out of 142 samples contained alpha-HCH residues at
levels of up to 0.03 mg/kg. Processed pork and poultry, sampled
during the period 1985-1987, contained alpha-HCH at levels of up to
3.2 (mean 0.2) and 0.1-2.0 (mean 0.8) mg/kg product, respectively (26
out of the 86 samples were positive). Of other processed meat
products, 631 samples were negative. Retail milk and dairy products
were analysed during the period 1984-1987, and 499 of the 849
samples contained alpha-HCH residues at a mean concentration of
0.01-0.03 mg/kg (highest level, 0.06 mg/kg). Samples of eel muscle
(1124 eels from 62 sites) were analysed during the period 1986-1987,
and mean concentrations were 0.001-0.03 mg/kg (highest level,
0.4 mg/kg). Peanut butter and vegetable oils were analysed during the
period 1985-1987, and 95 samples showed mean concentrations of <
0.01-0.03 mg/kg product (16 of the samples were positive) (HMSO,
1989).

The mean residue level of alpha-HCH in milk samples collected
during spring 1983 from 359 bulk transporters representing 16
counties, municipalities, and districts of Ontario was 5.3 µg/kg
butter fat. Alpha-HCH was found in over 90% of the samples (Frank et
al., 1985).

5.1.5 Terrestrial and aquatic organisms

5.1.5.1 Plants

Samples of three types of mosses and four types of lichens (in
total 13 samples) were collected in the Antarctic Peninsula (Graham
Land) in 1985, and alpha-HCH was detected in most of them at a mean
concentration of 0.4 (0.20-1.15) µg/kg (Bacci et al., 1986).

In a study by Gaggi et al. (1986), fallen leaves (at the end of
their natural life-cycle) and lichens were collected in 1984 at sites
near Florence and Siena, Italy, in a woodland hilly area away from
primary pollution sources. The leaves were from ten different species
of trees and two different lichen species were involved. The average
levels of alpha-HCH in leaves and lichen were 37 (16-61) µg/kg dry
weight and 27 (25-29) µg/kg dry weight, respectively. The same
authors reported that the levels of alpha-HCH in various plant species
collected in 14 countries were 0.5-2140 µg/kg dry weight.

5.1.5.2 Fish and mussels

Martin & Hartman (1985) analysed 60 fish samples from nine
locations in the north-central part of the USA and found
concentrations of 5-27 µg alpha-HCH/kg (wet weight) in 36% of the
samples. The frequency with which alpha-HCH was detected in fish from
the different rivers varied between 17 and 100%.

In a study by Saiki & Schmitt (1986), samples of three to five
adult bluegills (Lepomis macrochirus) and common carp (Cyprinus
carpio) were collected at eight sites in three rivers in California,
USA, in 1981. Alpha-HCH concentrations in carp of up to 0.036 mg/kg on
fat basis were reported, but the concentrations in bluegill were
lower.

Cowan (1981) studied the extent of pollution of Scottish coastal
waters by HCHs using Mytilus edulis as biological indicator. The
levels of alpha-HCH at the 118 sites sampled ranged from < 6 to
23 µg/kg dry weight.

The fish and shellfish sampling programme carried out by the
United Kingdom Ministry of Agriculture, Fisheries, and Food between
1977-1984 was directed mainly to areas around the coasts of England
and Wales. The levels of alpha-HCH, which varied between the
different fish and shellfish species and also between the collection
sites, ranged from < 0.001 (nd) to 0.06 mg/kg wet weight. The
concentration in fish muscle was < 0.001 mg/kg wet weight (Franklin,
1987).

The mean alpha-HCH concentration in the muscle of flounders
collected off the North Sea coast of Germany in 1986 was 2.5 µg/kg
(nd-5.0 µg/kg) (Umweltbundesamt, 1989). Bream collected from different
locations in the River Elbe (between Schnackenburg and the North Sea)
contained 0.007-0.066 mg alpha-HCH/kg in muscle tissue and

0.9-2.2 mg/kg in adipose tissue (Arbeitsgemeinschaft für die
Reinhaltung der Elbe, 1982), while the same species collected from 15
rivers and lakes in the Federal Republic of Germany contained (on a
fat basis) up to 468 µg per kg (Umweltbundesamt, 1989).

Freshwater fish from different rivers in the Federal Republic of
Germany were analysed during the period 1973-1981, and in the first
3-4 years the alpha-HCH levels were mainly between 0.01 and 0.02 mg/kg
fresh weight. However, a clear decrease then took place and most of
the samples were below 0.01 mg/kg fresh weight, with the exception of
certain types of fish such as the eel and fish from industrially
contaminated areas (Hildebrandt et al., 1986).

In 1981-1983, shellfish and molluscs collected in the Federal
Republic of Germany contained < 0.001-0.20 mg alpha-HCH/kg fresh
weight. Eels collected in the North Sea and Baltic Sea contained
alpha-HCH levels of 0.011 mg per kg and 0.033 mg/kg fresh weight,
respectively. Flounders and herrings caught in the North Sea contained
0.002 and 0.008 mg/kg fresh weight, respectively, but in the Baltic
Sea the levels were about twice as high (Hildebrandt et al., 1986).

5.1.5.3 Birds

An average alpha-HCH residue level of 0.05 mg/kg was found in 17
adult herons in 1964 (HMSO, 1969).

In a study by Sierra & Santiago (1987), alpha-HCH concentrations
were determined in 23 barn owls (Tyto alba Scop.) from Leon, Spain.
The mean levels (and range) in muscle, liver, fat, brain, and
kidneys (in total 91 samples) were 0.242 (0.019-0.591), 0.323
(0.009-0.830), 1.073 (0.691-1.499), 0.238 (0.007-0.676), and 0.710
(0.051-2.381) mg/kg (wet weight), respectively.

Faladysz & Szefer (1982) analysed adipose fat from seven species
of diving ducks at their winter quarters in the Southern Baltic.
Residues of alpha-HCH were found in all of the 37 specimens of
long-tailed duck at mean concentrations (on a fat basis) of 3.4
(0.17-18) and 1.5 (0.23-6) mg/kg for female and male ducks,
respectively.

5.1.5.4 Mammals

Skaftason & Johannesson (1979) analysed 24 samples of the fat of
reindeer living in an area of the eastern and south-eastern parts of
Iceland where the use of pesticides is negligible. Alpha-HCH was
found in all samples at a mean level of 70 ± 22 µg/kg. These results
are in agreement with those of Benson et al. (1973), who found an
average of 77.5 µg/kg in the subcutaneous fat of wild Idaho moose
living in a forest area where pesticides were used very restrictively.
Skaftason & Johannesson (1979) analysed samples of body fat from
10-year-old sheep in 1974 and found an average of 51 ± 12 µg/kg.

Norström et al. (1988) investigated the contamination by
organochlorine compounds of Canadian arctic and subarctic marine
ecosystems by analysing the adipose tissue and liver of polar bears
( Ursus maritimus; 6-20 animals per area) collected from 12 areas
between 1982 and 1984. There was a difference in tissue distribution;
liver contained only alpha-HCH, but 29% of the HCH in adipose tissue
was beta-HCH. Adipose tissue contained 0.3-0.87 mg alpha-HCH per kg on
a fat basis.

The mean concentrations of alpha-HCH in the kidney fat of roe
(86 samples) collected in five areas of Germany in 1985-1986 were
about 7-12 µg/kg fat, the maximum value being about 50 µg/kg fat
(Umweltbundesamt, 1989).

5.2 General population exposure

From the data presented in section 5.1 it is evident that food is
the main source of exposure of the general population to alpha-HCH.

5.2.1 Total-diet studies

In a total-diet study carried out in the United Kingdom during
1966-1985, food purchased in 21 towns throughout the country was
prepared by cooking. The study covered 20 to 24 food groups, and the
number of total-diet samples examined varied from 22 to 25 samples.
The calculated mean alpha-HCH residue levels in the total diet for the
periods 1966-1967, 1970-1971, 1974-1975, 1975-1977, 1979-1980, 1981,
and 1984-1985 were 0.003, 0.002, 0.002, 0.0015, 0.001, < 0.0005, and
< 0.0005 mg/kg, respectively (Egan & Hubbard, 1975; HMSO, 1982, 1986,
1989).

Gartrell et al. (1985a) conducted a study to determine the
dietary intake of pesticides in the USA in 1978-1979. The samples,
purchased from retail outlets, were representative of the diets of
adults in 20 cities, and consisted of about 120 individual food items.
The daily intake of alpha-HCH in 1977, 1978, and 1979 was 0.011,
0.009, and 0.010 µg/kg body weight, respectively. In a similar way,
samples were collected in 10 cities in 1978-1979 consisting of about
50 items of infant food and 110 items of toddler food. The daily
intake of alpha-HCH in 1977, 1978, and 1979 was, respectively, 0.031,
0.034, 0.033 µg/kg for infants and 0.025, 0.029, and 0.029 µg/kg body
weight for toddlers, respectively (Gartrell et al., 1985b).

Total-diet studies conducted in the USA by the FDA before 1982
were based on a "composite sample approach" regardless of the diet
involved. Later on they were based on dietary survey information and
allowed the "total diet" of the population to be represented by a
relatively small number of food items for a greater number of age-sex
groups. The daily intakes of alpha-HCH during 1982-1984 for the age
groups 6-11 months, 2 years, 14-16-year-old females, 14-16-year-old
males, 25-30-year-old females, 25-30-year-old males, 60-65-year-old

females, and 60-65-year-old males were 7.2, 16.1, 6.1, 7.3, 4.5, 5.9,
3.3, and 3.7 ng/kg body weight, respectively (Gunderson, 1988).

In a total-diet study in the Netherlands in 1977, the average
concentration of alpha-HCH in 100 samples was 0.01 mg/kg on a fat
basis. The highest level was 0.05 mg/kg (Greve & van Hulst, 1977).

5.2.2 Air

Guicherit & Schulting (1985) measured the atmospheric
concentration of alpha-HCH in the Netherlands and calculated an
average daily intake by inhalation for a 70-kg person of 5 ng. The
equivalent value for the Federal Republic of Germany was calculated to
be 32 ng, which is about 1% of the total daily intake via the various
routes (Hildebrandt et al., 1986).

5.2.3 Concentrations in human samples

Alpha-HCH concentrations in human samples are a good indication
of the total exposure of the general population.

5.2.3.1 Blood

Blood samples of Dutch citizens analysed in 1978, 1980, 1981, and
1982 (70, 48, 127, and 54 samples, respectively), contained less than
0.1 µg alpha-HCH/litre (Greve & Wegman, 1985). Blok et al. (1984)
analysed the blood of 65 healthy volunteers in the Netherlands (34
female and 31 male) and detected alpha-HCH in less than one third of
the samples. The median concentration for both groups was below the
detection limit (0.1 µg per litre), but levels of up to 0.4 µg/litre
were measured.

Polishuk et al. (1970) studied the presence of alpha-HCH in the
blood of 24 pregnant women and 23 infants living in Israel. The
mean concentration was 0.6 ± 0.3 µg per litre in the women and 0.5 ±
0.3 µg/litre in the infants.

In 1975, Reiner et al. (1977) analysed the serum and plasma of 82
women and 65 men (with an average age of 42) living in a town in
Yugoslavia. In 57 of the 147 samples, alpha-HCH was found at a mean
concentration of 3.3 ± 0.5 µg/litre (range, 0.1-15.0 µg/litre).
Similar values were found in other parts of the country in 1976-1979
(Krauthacker et al., 1980).

The median concentration of alpha-HCH in whole blood of 118
people in the Federal Republic of Germany was reported to be
0.98 µg/litre (range, nd-2.06) (Bertram et al., 1980).

5.2.3.2 Adipose tissue

The alpha-HCH concentrations of 567 samples of adipose tissues of
Dutch citizens analysed during 1968-1983 varied from < 0.01 to
0.1 mg/kg (on a fat basis). The highest levels occurred during the
period 1968-1976 (Greve & van Harten, 1983; Greve & Wegman, 1985).

In a study by Niessen et al. (1984), specimens of subcutaneous
adipose tissue from 48 infants (34 under the age of 1 year, 14 in
their second year of life) were examined during 1982-1983 in the
Federal Republic of Germany. The average concentration of alpha-HCH
was 0.01 mg/kg fat (range, nd-0.02 mg/kg). The average concentration
was highest (0.02 mg/kg fat) for the age-range 0-6 weeks. Bertram et
al. (1980) found a median concentration of 0.03 mg/kg fat (range,
nd-0.35) in 72 samples of adipose tissue from people in the Federal
Republic of Germany. Hildebrandt et al. (1986) summarized the results
of nine studies carried out in the Federal Republic of Germany during
1969-1983. The mean alpha-HCH concentrations (568 samples) ranged
from 0.01 to 0.03 mg/kg fat.

Mes et al. (1982) analysed 99 samples of adipose tissue from
autopsies of accident victims from different areas of Canada. Nearly
all the samples (97%) contained alpha-HCH, the average concentration
of which was 0.004 mg/kg wet weight (range, 0.001-0.043 mg/kg).

In 1974, 360 samples of adipose tissue were collected in eight
regions of Japan and the mean level of alpha-HCH was 0.031 mg/kg
tissue (Takabatake, 1978).

Twenty-nine samples of adipose tissue were taken at necropsy and
24 at surgery in the Poznan district of Poland and compared with 100
samples from residents of the Warsaw area. In Poznan the mean
concentration of alpha-HCH was 0.013 ± 0.033 mg/kg, while in Warsaw it
was 0.008 ± 0.001 mg/kg (Szymczynski et al., 1986).

5.2.3.3 Breast milk

Breast milk is a major route for the elimination of
organochlorine pesticides in women. In a Swedish study, the levels of
alpha-HCH in breast milk were found to be related to the dietary
habit. Levels in lacto-vegetarians were lower than those in women
eating a mixed diet, and these were lower than those found in mothers
using a mixed diet that regularly included fatty fish from the Baltic
(Noren, 1983).

In a study by Fooken & Butte (1987), the variation of residue
levels in breast milk during lactation was investigated in five women
(aged 23-36) in the Federal Republic of Germany. Alpha-HCH
concentrations of up to 0.009 mg/kg fat were measured, and no
essential changes in residue level occurred over the lactation period.

Residues of alpha-HCH in breast milk during the periods,
1974-1975 and 1979-1980 in the Federal Republic of Germany were
reported to be 0.03 and 0.02 mg/kg milk on a fat basis, respectively
(Anon., 1984).

In the Federal Republic of Germany, more than 7100 samples of
breast milk were analysed from 1969-1984. These studies were carried
out by 20 authors, and the results were summarized by Hildebrandt et
al. (1986). The mean concentrations of alpha-HCH ranged from
0.01-0.04 mg/kg on a fat basis. In one case a mean concentration of
0.21 mg/kg was found in 320 samples. During the period investigated, a
slow decrease in the mean concentration of alpha-HCH was observed. The
average concentration in breast milk in the same country (2709
samples) in 1979-1981 was 0.024 mg/kg on a fat basis (Fooken & Butte,
1987). In 1981-1983, 132 samples of breast milk were analysed and the
average level was 0.001 mg alpha-HCH/kg milk fat (Cetinkaya et al.,
1984).

Tuinstra (1971) analysed 36 individual samples of breast milk,
collected in 1969, from young mothers (18-32 years of age) living in
the Netherlands. A median alpha-HCH concentration of 0.01 mg/kg milk
(on a fat basis) was found (range, nd-0.04). When 278 samples of
breast milk, collected in 11 maternity centres in the Netherlands,
were analysed for the presence of alpha-HCH, the median alpha-HCH
concentration was < 0.01 mg/kg (on a fat basis) (Greve & Wegman,
1985).

Vukavic et al. (1986) measured the alpha-HCH concentration in 59
samples of colostrum collected during autumn 1982 (26 samples) and
spring 1983 (33 samples) in Yugoslavia from healthy nursing mothers on
the third day after delivery. The alpha-HCH levels were significantly
lower in the autumn than in the spring (mean concentrations of 0.49 ±
0.09 and 1.50 ± 0.26 µg/litre whole colostrum, respectively).

Mes et al. (1986) studied 210 breast milk samples from five
different regions of Canada and measured a mean alpha-HCH
concentration of 7 µg/kg (on a fat basis). Davies & Mes (1987)
studied 18 breast milk samples from Canadian, Indian, and Inuit
mothers in Canada, whose fish consumption was comparable to the
national level. The level of alpha-HCH in milk fat of the indigenous
population was 5 µg/kg, which was the same value as that obtained from
a national survey.

6. KINETICS AND METABOLISM

6.1 Absorption and elimination

The intestinal absorption rate for alpha-HCH was 97.4% after the
administration of an HCH mixture to male rats (Albro & Thomas, 1974).

The total excretion in rats after a single intraperitoneal (ip)
³⁶Cl-labelled alpha-HCH dose of 200 mg/kg body weight was 80% of the
dose in the urine and 20% in the faeces (Koransky et al., 1963;
Koransky et al., 1964; Noack et al., 1975). In a study in rats with
³⁶Cl-labelled alpha-HCH, a low excretion rate was found. ³⁶Cl was
detected in the excreta up to 40 days after a single ip dose (Koransky
et al., 1963). During continued dosing alpha-HCH was observed to
stimulate its own degradation (Noack et al., 1975). The decrease in
rat liver alpha-HCH levels after an initial increase, observed by
Eichler et al. (1983), was assumed to be due to this effect.

When ¹⁴C-labelled alpha-HCH was administered intraperitoneally
to male mice (ddY-strain, 4 weeks old) at a dose level of 22 µg, the
average percentage of urinary excretion of radioactivity in 3 days was
37% (Kurihara & Nakajima, 1974).

6.2 Distribution

One day after an ip injection of a mixture of ¹⁴C- and
³⁶Cl-labelled alpha-HCH into rats (200 mg/kg body weight in rapeseed
oil), the highest level of radioactivity was found in fat, skin, and
bones plus muscles (18.2, 13.1, and 11.9%, respectively, after 4
days). Much lower levels were found in other organs or tissues (up to
1% in liver and kidneys and 0.28% in the central nervous system. In
the faeces and urine, 3.9 and 7.9%, respectively, were found after 4
days (Koransky et al., 1963). In other studies with rats, higher
concentrations were found in liver, kidneys, body fat, brain, and
muscle (Portig & Vohland, 1983; Kuiper et al., 1985). In a 90-day
study in rats, marked deposition of alpha-HCH was found in renal fat;
the concentrations exceeded those obtained in a similar study on
beta-HCH (Greve & van Hulst, 1980; Kuiper et al., 1985). In lactating
rats given a single oral dose 5 days after birth, the alpha-HCH
concentrations in the livers of the sucklings were twice as high as
those observed in the livers of the mothers (Wittich &
Schulte-Hermann, 1977).

Vohland et al. (1981) studied the distribution of alpha-HCH in
the brain and depot fat of rats after the administration of 200 mg/kg
body weight by gavage. With an average blood concentration of
1.5 µg/litre, the brain to blood, and depot fat to blood ratios were
120:1 and 397:1, respectively, whereas with a blood concentration of
17.7 mg/litre the ratios were 5:1 and 82:1, respectively.

Nagasaki (1973) orally administered alpha-HCH to male mice at
concentrations of 100, 250 or 500 mg/kg for 24 weeks, and found high
residual levels of this isomer in liver and adipose tissue. Similarly,
Macholz et al. (1986) reported that a 30-day administration of
alpha-HCH to rats resulted in high residues of this isomer in fat,
kidneys, and adrenal tissue.

In the brain, alpha-HCH is stored preferentially in the white
matter (Stein et al., 1980; Portig et al., 1989).

6.3 Metabolic transformation

6.3.1 Rat

When Sprague-Dawley weanling female rats were administered 2 mg
alpha-HCH/rat per day in peanut oil for 7 days, the alpha-HCH was
metabolized to 2,4,6- and 2,4,5-trichlorophenol, with an excretion
ratio of 2,4,6- to 2,4,5-trichlorophenol of 1.3:1. This study also
indicated that pre-treatment with alpha-HCH alters the metabolism of
lindane in rats (Freal & Chadwick, 1973).

The biotransformation of alpha-HCH in rats involves
dechlorination (Kraus, 1975). The dose-dependent decrease in liver
glutathione concentrations indicates the formation of a glutathione
conjugate in this organ (Noack & Portig, 1973; Portig et al., 1973;
Kraus, 1975). Such a decrease does not occur in the brain or kidneys
(Noack & Portig, 1973).

The major urinary metabolite in rats is 2,4,6-trichlorophenol, a
compound reported by IARC (1987) to be carcinogenic for animals
(Portig et al., 1973; Stein & Portig, 1976; Stein et al., 1977). Other
metabolites that have been identified are 1,2,4-trichlorophenol,
2,3,4-trichlorophenol, 2,4,5-trichlorophenol, 2,3,4,5-
tetrachlorophenol, and 2,3,4,6-tetrachlorophenol (Noack et al., 1975;
Stein et al., 1977; Macholz et al., 1982). In addition,
chlorothiophenols (not specified) have been detected, and
1,3,4,5,6-pentachlorocyclohex-1-ene has been identified in the kidneys
of rats (Macholz et al., 1983).

Artigas et al. (1988) have identified several lindane metabolites
(tetra-, penta-, and hexachlorocyclohexenes, and tetra- and
pentachlorobenzenes) in rat brain homogenates by gas
chromatography-mass spectrometry. Male Wistar rats were orally
administered 30 mg alpha-HCH/kg and were sacrificed 5 h later. The
cerebella of the animals were analysed and the following metabolites
were found: 3.6/4.5-PCCH, 3.5/4.6-PCCH, HCCH, pentachlorobenzene, and
HCB. HCCH was the major metabolite (about 100 µg per kg) while levels
of the other metabolites were mainly below 5 µg/kg. Alpha-HCH was
present at 17.2 mg/kg tissue. This study showed that the HCH isomers
are cleared from the brain via different metabolic pathways.

Isomerization of alpha-HCH to lindane did not occur after
repeated dosage (Eichler et al., 1983).

6.3.2 Bird

In a model 4-week feeding study on poultry using four HCH
isomers, the rate of degradation of the individual HCH isomers in
broilers followed the order: delta > gamma > alpha > beta.
Biotransformation (to one or more of the other HCH isomers) was not
detected (Szokolay et al., 1977b).

In a study by Foster & Saha (1978) on the in vitro metabolism
of alpha-HCH in chicken livers, the first metabolite was identified as
an isomer of pentachlorocyclohexane.

6.3.3 Human

When Engst et al. (1978) analysed the urine of occupationally
exposed workers (apparently to technical-grade HCH in manufacturing
processes), they found, apart from alpha-, beta-, gamma-, and
delta-HCH, traces of hexa- and pentachlorobenzene, gamma- and
delta-pentachlorocyclohexane, pentachlorophenol, 2,3,4,5-, 2,3,4,6-,
and 2,3,5,6-tetrachlorophenol, and several trichlorophenols, as well
as the glucuronides of several of these metabolites. The
pentachlorocyclohexenes, tetrachlorophenol, hexachlorobenzene, and
pentachlorophenol were also identified in the blood.

6.4 Retention and biological half-life

The half-life for the clearance of alpha-HCH from depot fat was
found to be 6.9 days in female rats and 1.6 days in male rats (Stein
et al., 1980; Portig, 1983).

Vohland et al. (1981) and Portig & Vohland (1983) studied the
kinetics of alpha-HCH in Wistar rats, and observed that, after a
single oral dose of 200 mg/kg body weight, the approximate half-life
in females for the elimination from brain was 6 days.

The retention of alpha-HCH in rat brain after a single dose is
greater than that of beta- and gamma-HCH (Stein et al., 1980).

7. EFFECTS ON LABORATORY MAMMALS AND IN VITRO TEST SYSTEMS

7.1 Single exposure

7.1.1 Acute toxicity

In mice oral LD₅₀ values have been found to range from 1000 to
4000 mg/kg body weight, depending on the vehicule, while in rats
values of 500-4674 mg/kg body weight have been obtained. Riemschneider
(1949) determined a LD₅₀ (oral intubation in olive oil) for rats of
1500 mg/kg body weight. The signs of poisoning were those of nervous
system stimulation: excitation, hunched posture, rough fur, dyspnoea,
anorexia, tremors, convulsions, and cramps (Hoffmann, 1983; WHO,
1986).

7.2 Short-term exposure

7.2.1 Oral

In a 90-day study on rats carried out with dose levels of 0, 2,
10, 50, or 250 mg alpha-HCH/kg diet, reductions in white blood cell
count were noted in several groups of animals. Growth was decreased
at 250 mg/kg diet, and at this dose level the number of erythrocytes
and protein excretion in the urine were elevated in female animals. At
levels of 50 and 250 mg/kg, the activities of liver
amino-pyrine- N-demethylase and aniline hydroxylase were increased
while those of blood aspartate aminotransferase (ASAT) and creatine
phosphokinase were decreased. Liver weights were increased at dose
levels of 10, 50, and 250 mg/kg. Enlargement of liver parenchyma cells
(with a foamy/hyaline appearance of the cytoplasm) and accentuation of
the plasmalemma, indicative of proliferation of smooth endoplasmatic
reticulum (SER), occurred at levels of 50 and 250 mg/kg. At the
250-mg/kg level, there were increases in the relative weights of
heart, kidneys, and adrenals. In addition, serum levels of
immunoglobulins G and M showed a decrease at 50 and 250 mg/kg diet
(Kuiper et al., 1985).

Macholz et al. (1986) reported that the administration of 1000 mg
alpha-HCH/kg to rats for 30 days resulted in growth retardation and
liver mass increase. High residue levels of alpha-HCH were identified
in fat, kidneys, and adrenal tissue.

7.2.2 Other routes

7.2.2.1 Intravenous

In a study by van Asperen (1954), groups of 12-15 male and female
albino mice (8-10 weeks of age) were given an intravenous injection of
alpha-HCH (in peanut oil). The dose levels were 480 or 960 µg/mouse
(equivalent to approximately 32 and 64 mg/kg body weight,
respectively). No deaths occurred within 7 days.

7.2.2.2 Subcutaneous

Groups of 13-21 male and female albino mice (8-10 weeks of age)
were given a subcutaneous injection of alpha-HCH at dose levels
ranging from 3 to 20 mg/animal (equivalent to approximately 200 to
1330 mg/kg body weight, respectively). With doses of up to 4 mg, no
death occurred within 7 days, but with 4.5 mg, 8 mg, and 20 mg, 8, 25,
and 90%, respectively, of the animals died (van Asperen, 1954).

7.3 Skin and eye irritation; sensitization

No data on skin and eye irritation or sensitization have been
reported.

7.4 Long-term exposure

7.4.1 Rat oral study

When groups of 10 female and 10 male weanling Wistar rats were
administered diets containing 0, 10, 50, 100, or 800 mg alpha-HCH/kg
diet (in corn oil) for 107 weeks, the highest dose level resulted in
growth retardation, increased mortality, and slight kidney damage.
With dose levels of 100 or 800 mg/kg, liver enlargement and
histo-pathological changes in the liver were found. However, there
were no liver changes at 50 mg/kg diet (Fitzhugh et al., 1950).

7.5 Reproduction, embyrotoxicity, and teratogenicity

No information on reproduction, embryotoxicity, or teratogenicity
is available.

7.6 Mutagenicity and related end-points

Alpha-HCH did not induce mutations in Salmonella typhimurium
test strains TA98, TA100, TA1535 or TA1537 either with or without rat
liver metabolic activation (Lawlor & Haworth, 1979). A test for point
mutations in Saccharomyces cerevisiae XV 185 14 C was also negative
(Shahin & von Borstel, 1977). In addition, the compound produced no
mutations in Allium cepa roots (Nybom & Knutsson, 1947). A test for
unscheduled DNA synthesis in rat hepatocytes in vitro produced an
equivocal result (Althaus et al., 1982).

A mutagen test strain of Bacillus subtilis (TKJ5211) showed a
higher sensitivity for hisw⁺ reversion than the parental strain
HA101 when treated with UV and UV-mimetic chemicals. However, a
negative result was obtained when alpha-HCH dissolved in DMSO was used
at a dose level of 5 mg/ml (Tanooka, 1977).

A DNA repair test was carried out with stationary-phase cultures
of B. subtilis HLL3g and HJ-15 strains in which the size of growth
inhibition zones of repair-proficient and repair-deficient cells for
vegetative cells and spores was determined. There was no effect at a
dose level of 5 mg alpha-HCH (in benzene) per ml (Tanooka, 1977).

The available data are inadequate to make an assessment of the
mutagenic potential.

7.7 Carcinogenicity

Appraisal

The reported studies on the carcinogen effects of alpha-HCH on
mice and rats have some short-comings. In most cases, very high dose
levels were tested. Nevertheless, it is clear from the results that
alpha-HCH, at high dose levels, produces nodular hyperplasia and
hepatocellular carcinomas in mice (the incidence varying according to
the strain) and also in rats (low incidence), but only at higher dose
levels.

The results of the studies on initiation-promotion and mode of
action indicate that the neoplastic response observed with alpha-HCH
is most likely due to a non-genotoxic mechanism.

7.7.1 Mouse

When 20 male ICR/JCL mice (aged 5 weeks) were administered a diet
containing 600 mg alpha-HCH/kg diet for 26 weeks, increased liver
weight was observed. In all treated mice there were liver tumours,
which were characterized histologically as benign tumours and
malignant tumours with atypical liver cells. Unfortunately,
insufficient details were reported (Goto et al., 1972a,b).

In a study by Hanada et al. (1973), 6-week-old DD mice (10-11 of
each sex per group) were given diets containing 0, 100, 300, or 600 mg
alpha-HCH/kg diet for 32 weeks, followed by a control diet for 5-6
weeks. The control group consisted of 20 female and 21 male animals.
During the experiment several animals died. The numbers of hepatomas
in the four groups surviving for 36-38 weeks were 0/29 (control), 1/16
(100 mg/kg), 9/10 (300 mg/kg), and 13/15 (600 mg/kg).
Alpha-fetoprotein was not detected in the serum of animals with
hepatomas.

When 8-week-old male DD mice, divided into groups of 20 or 38
animals, were fed a diet containing 0, 100, 250, or 500 mg
alpha-HCH/kg for 24 weeks, the two highest dose levels induced an
increase in liver weight. At the four respective dose levels, the
incidence of nodules classified as nodular hyperplasia was 0/20, 0/20,
30/38 (79%), and 20/20 (100%) and that of hepatocellular carcinoma was
0/20, 0/20, 10/38 (26%), and 17/20 (85%) (Ito et al., 1973b).

Following the oral administration of 100, 250 or 500 mg
alpha-HCH/kg to male DD mice for 24 weeks, hepatocellular tumours were
found in all mice treated with 500 mg/kg and in 17 of the 20 mice that
received 250 mg/kg (Nagasaki, 1973).

Nagasaki et al. (1975) studied the tumorigenic effects of a diet
containing 0 or 500 mg alpha-HCH/kg, fed for 24 weeks to groups of
male and female DDY, ICR, DBA/2, C57BL/6, and C3H/He mice (13-29 of
each sex per group), male Wistar rats, and male golden Syrian
hamsters. It was found that alpha-HCH induced liver tumours in male
and female mice but not in rats and hamsters. The histological changes
in the liver of mice were much greater than those induced in rats and
hamsters. Male animals were more susceptible to the tumorigenic
action (i.e. liver nodules) than females. Among the different strains
of mice, a difference in susceptibility was observed. The occurrence
of liver nodules varied from 16.7 to 100% and the incidence of
hepatocellular carcinomas varied from none to 65%. The DDY mouse
strain was the most sensitive and the C57BL/6 the least sensitive
strain.

Ito et al. (1976) studied the reversibility of liver tumours
induced by alpha-HCH (99.0%). Male 8-week-old DDY mice were fed a
diet containing 0 or 500 mg/kg for 16, 20, 24, and 35 weeks and then
fed a basal diet without alpha-HCH for 4, 8, and 12, or 4, 8, 12, 16,
24, and 36 weeks. In total 341 mice were used, of which 21 were fed
the compound for 16 weeks. A total of 300 mice were fed the diet with
alpha-HCH for 20 or more weeks and 20 control mice were fed the basal
diet for 72 weeks. At the various intervals indicated, 12-20 mice were
killed. The incidence of liver tumours increased progressively during
continuous administration of alpha-HCH, but when its administration
was discontinued some tumours disappeared. After 24 weeks of
administration most tumours were nodular hyperplasias with only a few
well-differentiated hepatocellular carcinomas. However, 60 or 72
weeks after the beginning of the study most of the liver tumours were
hepatocellular carcinomas. The findings suggested that nodular
hyperplasia was usually reversible.

Two groups of male HPBC57B1 black mice (6-9 weeks old) were fed
a diet containing 500 mg/kg alpha-HCH (99.8%) per diet, 48 mice being
used as controls and 75 mice being administered alpha-HCH. From each
group, 4-9 mice were killed at 1, 3, 4, 8, 14, 21, 30, 33, 44, and 50
weeks after the initiation of treatment. Progressive liver
enlargement was first noticed at 3 weeks, hepatic nodules at 21 weeks,
and emaciation at 30 weeks. Histopathological liver alterations
included hypertrophy of centrolobular hepatocytes first seen at 1 week
and the merging of adjacent megalocytic zones at 3 weeks. At 21
weeks, adenomas were seen in two out of seven mice, at 30 weeks in
seven out of eight mice, and at 33, 44, and 50 weeks in all the mice
studied. Under the condition of this study, neither hepatocellular
carcinomas nor metastases in the lungs were detected (Tryphonas &
Iverson, 1983).

7.7.2 Rat

When groups of 10 male and 10 female weanling Wistar rats were
fed throughout their life on diets containing 10, 50, 100, or 800 mg
alpha-HCH (> 98% pure) per kg, no increase in tumour incidence was
found. However, only a limited number of organs were examined
microscopically (Fitzhugh et al., 1950).

In a study by Ito et al. (1975), male Wistar rats (5-8-weeks old)
were divided into seven groups and administered alpha-HCH diets
containing 0, 500 (two groups), 1000 (three groups), or 1500 mg
alpha-HCH/kg diet. The duration of the treatment for the different
groups was 72 weeks for the controls, 24 or 48 weeks at 500 mg/kg, 24,
48, or 72 weeks at 1000 mg/kg, and 72 weeks at 1500 mg/kg. In the
liver, oval cells and bile duct cell proliferation were found in the
groups fed 1000 or 1500 mg/kg after 48 and 72 weeks. Cell hypertrophy
was found in all the groups, the increase in severity depending on the
dose level and the duration of administration. In the two groups fed
500 mg/kg and the group fed 1000 mg/kg for 24 weeks no nodular
hyperplasia or hepatocellular carcinomas were found. Nodular
hyperplasia developed in the groups fed 1000 mg/kg (48 and 72 weeks)
or 1500 mg/kg (72 weeks) in 42, 76, and 77% of the animals,
respectively. Hepatocellular carcinomas were found only in the groups
fed 1000 or 1500 mg/kg for 72 weeks (1/16 and 3/13 animals,
respectively).

In a series of studies, an oral dose of 20 mg/kg body weight was
administered daily to female rats during periods of 4.5, 13.5, or 23.5
months. Liver enzyme induction was found at all intervals, white foci
and nodules were present after 13.5 months, and one animal had a
hepatocellular carcinoma after 23.5 months (Schulte-Hermann &
Parzefall, 1981). The value of this study was reduced by the very low
number of animals (4-6 per group) used at each interval.

7.7.3 Initiation-promotion

In a study on 8-week-old white male mice (25-30 per group) of
strain DD, the influence of alpha-HCH on tumour induction by
polychlorinated biphenyls (PCBs) was tested and vice versa. Whereas
500 mg PCB/kg diet induced nodular hyperplasia and hepatocellular
carcinomas in the liver of male mice after 32 weeks, exposure to
alpha-HCH at dose levels of 50, 100, or 250 mg/kg diet, only resulted
in both type of tumours at the highest dose level. The incidence of
nodular hyperplasia was 23/30 (77%) and that of hepatocellular
carcinoma was 8/30 (27%). However, 50 or 100 mg alpha-HCH/kg diet, in
combination with 250 mg PCB per kg diet (PCB alone did not induce
tumours), induced nodular hyperplasia (approximately 30%) and
hepatocellular carcinoma (approximately 5%). It seems that PCBs
promote the induction of liver tumours by alpha-HCH (Ito et al.,
1973a).

In studies on rats, alpha-HCH showed a tumour-promoting action
towards the hepatocarcinogenic effects of aflatoxin B1,
diethylnitrosamine, and nitrosomorpholine (Schulte-Hermann &
Parzefall, 1981; Schulte-Hermann et al., 1981; Angsubhakorn et al.,
1981). In one test, alpha-HCH produced only a slight liver
tumour-promoting effect in rats after initiation with
N-nitrosodiethylamine (Ito et al., 1983). However, in another study
on the same species the compound had an inhibitory effect on the
hepa-tocarcinogenic action of 3-methyl-4-dimethylaminoazobenzene and
DL-ethionine (Thamavit et al., 1974).

Nagasaki et al. (1975) studied the influence of
3-methylcholanthrene, 1-naphthyl isothiocyanate, and
p-hydroxypropiophenone on the induction of liver tumours by
alpha-HCH. Eight groups of 24 mice received a diet containing either
500 mg alpha-HCH/kg diet in combination with 67 mg
methylcholanthrene/kg, 600 mg 1-naphthyl isothiocyanate/kg or 1000 mg
p-hydroxypropiophenone/kg or just one of the four compounds. A
control group with the basal diet was also used. The induction of
mouse liver tumours by alpha-HCH was not inhibited by the concomitant
feeding of 1-naphthyl isothiocyanate or p-hydroxypropiophenone.
However, 3-methylcholanthrene slightly inhibited their induction by
alpha-HCH.

In a study by Schröter et al. (1987), the tumour-initiating
activity of alpha-HCH was studied by examining for phenotypically
altered foci in female Wistar rats. Groups of three to eight rats
were used and, after removing the median and right liver lobes, 200 mg
alpha-HCH/kg body weight was administered followed by phenobarbital at
50 mg/kg body weight per day for 15 weeks. Liver foci were identified
by means of the gamma-glutamyltransferase (GGT) reaction and by
morphological alterations. No evidence of initiating activity was
found. In another part of the study, the promoting activity was
investigated. A single dose of N-nitrosomorpholine (250 mg/kg body
weight by gavage) was followed by the administration of 0.1, 0.5, 2.0,
7.0, or 20.0 mg alpha-HCH/kg body weight per day for 4, 15, and 20
weeks. The criteria used were growth and phenotypic changes of foci
as end-points. It was concluded from the study that alpha-HCH is a
tumour promotor. Both the number and size of altered foci were
enhanced by alpha-HCH doses of 2 mg/kg or more. The tumour-promoting
action was generally associated with liver enlargement and induction
of monooxygenases or other specific enzymes.

Schulte-Hermann et al. (1983) carried out three experiments with
Han-Wistar rats using, in experiment 1, 39 female rats (8-24 months
old) and, in experiments 2 and 3, 41 male (2 years old) rats.
Alpha-HCH (200 mg/kg in corn oil) was administered orally as a single
dose, while the control group received only corn oil. Beginning 25 h
after the dosing, ³H-thymidine was injected intravenously five times
at intervals of 6 h (experiment 2) or 8 h (experiment 3) , and the
animals were killed 18 (experiment 2) or 3 h (experiment 3) after the

last dose of ³H-thymidine. The effect of age on the incidence of
spontaneous foci was studied in experiment 1. Foci of putative
preneoplastic cells were detected in the livers of untreated rats of
both sexes, especially at 1 and 2 years of age. These foci exhibited
markers similar to those of their counterparts in carcinogen-treated
rats, such as cytoplasmic basophilia, clearness of cytoplasm, or
expression of gamma-glutamyl transferase. Rates of DNA synthesis in
foci were higher than in normal liver cells and were increased by
single doses of liver mitogens assumed to promote liver tumour
development. Thus cells in the spontaneous foci appeared to possess a
defect in the growth control, rendering them more susceptible to
endogenous and exogenous growth stimuli.

The incorporation of orally administered radiolabelled thymidine
into liver DNA was determined in SIV-50-SD rats 24 h after a single
oral gavage dose of 2.9, 29.1, 58.2, or 291 mg alpha-HCH/kg. Alpha-HCH
was found to stimulate liver DNA synthesis at 58.2 mg/kg (Büsser &
Lutz, 1987).

7.7.4 Mode of action

Sagelsdorff et al. (1983) studied the relevance to the
carcinogenic action of alpha-HCH of covalent binding to mouse liver
DNA. Three strains of mice were used (NMRI, CF₁, and C6B3F₁), and
alpha-HCH was administered by oral gavage and ¹⁴C-thymidine by the
intraperitoneal route. In all three strains, a similar low covalent
binding index or DNA damage/dose (values ranging from 0.17-0.28) was
found. There was no quantitative correlation with the carcinogenicity
potency of alpha-HCH.

Iverson et al. (1984) studied the ability of alpha-HCH to bind to
macromolecules from male HPB black mouse liver. In vivo and in
vitro binding studies with ¹⁴C-alpha-HCH and hepatic microsomes
from untreated and phenobarbital-pretreated mice showed no
preferential binding of alpha-HCH to protein or DNA. The results
suggest that the neo-plastic response observed with alpha-HCH results
from a non-genotoxic mechanism.

7.8 Special studies

7.8.1 Effect on liver enzymes

After a single oral administration to female rats of 5 mg
alpha-HCH/kg body weight or more the rate of aminopyrine demethylation
and the liver DNA content were both increased, but at 2 mg/kg body
weight these effects did not occur (Schulte-Hermann et al., 1974). In
a further study, the liver cytochrome P450 concentration in male rats
after a single oral administration was elevated at all tested dose
levels, 25 mg/kg body weight being the lowest (Seifart & Buchar,
1978). After alpha-HCH was given to male rats at dose levels of 5, 10,

20, 50, or 200 mg/kg feed for 2 weeks, aniline hydroxylase and
aminopyrine demethylase activities were increased at all dose levels
(den Tonkelaar et al., 1981).

7.8.2 Neurotoxicity

Appraisal

Alpha-HCH has been shown to have no effect on motor nerve
conduction velocity or the fronto-occipital EEG in rats fed 1000 mg
alpha-HCH/kg diet for 30 days. This isomer is a mild antagonist of
pentylenetetrazol-induced convulsions but increases the tonic/clonic
activity and the lethality of picro-toxin when administered
intraperitoneally to mice. It decreases the accumulation of
cerebellar cyclic GMP and prohibits the increase of cGMP caused by
gamma-HCH in mouse brain. Alpha-HCH has been demonstrated to inhibit
GABA-mediated chloride ion uptake in mouse brain, and this effect is
believed to play a primary role in the CNS action of this isomer.

In a study by Vohland et al. (1981), alpha-HCH did not give rise
in brain tissue to appreciable quantities of hydrophobic metabolites
such as 2,4,6-trichlorophenol. It had a weak protecting action
against convulsions induced by pentylenetetrazole (PTZ). The intensity
and duration of the PTZ-antagonistic effects after a single oral dose
were related to the alpha-HCH content of the brain.

In a 30-day study on groups of 15 male Wistar rats fed alpha-HCH
at levels of up to 1000 mg/kg diet, there was no effect on the
fronto-occipital electroencephalogram or on the motor conduction
velocity of the tail nerve (Müller et al., 1981).

The effect of alpha-HCH on body temperature, food intake, and
body weight was studied in Wistar rats (eight males and eight females)
given a single 30-mg/kg oral dose of alpha-HCH in olive oil. Controls
received only olive oil. Alpha-HCH treatment induced no significant
decrease in core temperature 5 h after treatment, and no decrease in
food intake or growth was observed (Camon et al., 1988).

Fishman & Gianutsos (1987) studied the effects of an
intraperitoneal injection of alpha-HCH (99.0%) in corn oil
(80-480 mg/kg body weight) on the accumulation of cerebellar cyclic
GMP in male CD-1 mice. Alpha-HCH decreased the accumulation of
cerebellar cyclic GMP and also prevented the increase in cyclic GMP
resulting from lindane treatment. Furthermore, alpha-HCH inhibited
the binding of ³H-TBOB (a ligand for the GABA-A-receptor-linked
chloride channel) in mouse cerebellum.

Fishman & Gianutsos (1988) compared the CNS-related
pharmacological and biochemical effects of gamma-HCH and the
non-convulsant isomer alpha-HCH. The studies were carried out on male
CD-1 mice injected intraperitoneally with a single alpha-HCH (in corn

oil) dose of 80-400 mg/kg body weight. Alpha-HCH inhibited the
myoclonic jerk and tonic/clonic activity of PTZ but increased the
tonic/clonic activity and lethality of picrotoxin (PIC) (PTZ and PIC
were given as a single ip injection of 50 mg/kg and 20 mg/kg body
weight, respectively). The highest dose of alpha-HCH caused a
significant decrease in motor activity. Gamma-HCH inhibited the
binding of ³H-TBOB to mouse whole brain membranes. Furthermore, this
isomer is a weak inhibitor of GABA-stimulated uptake of ³⁶wCl into
mouse brain neurosynaptosome preparations in vitro. The
non-seizure-inducing alpha-HCH has biochemical and pharmacological
effects in the CNS which differ from those of the gamma-HCH.

Matsumoto et al. (1988) provided evidence that all HCH isomers
are capable of inhibiting GABA-A-mediated chloride channels in the
brain, the relative potency being alpha = gamma > delta > beta.
Alpha-HCH was also found to be a potent inhibitor of the
batrachotoxin-stimulated action potential flux of sodium ions in N18
neuroblastoma cell cultures (Shain et al., 1987).

8. EFFECTS ON HUMANS

8.1 Acute toxicity - poisoning incidents

Several cases of acute poisoning by technical-grade HCH,
resulting either from accidents or occupational exposure, have been
described (WHO, 1991). Although alpha-HCH constitutes 65-70% of the
technical product, it is likely that the most acutely toxic component,
i.e. gamma-HCH, played the major role in these incidents. These cases
cannot, therefore, assist in the evaluation of alpha-HCH.

8.2 General population

No specific studies relating to alpha-HCH are available.

A study comparing liver cancer deaths in the USA and the
"domestic disappearance" of organochlorine pesticides revealed that in
1962, 18 and 15 years after the introduction of DDT and
technical-grade HCH, respectively (when an increase in primary liver
cancer due to the organochlorines would be manifest), the number of
cases of primary liver cancer as a percentage of the total number of
liver cancer deaths began a gradual and steady decline (from 61.3% in
1962 to 56.9% in 1972). The death rate (per 100 000 per year) due to
primary liver cancer declined from 3.46 to 3.18 during this period
(Deichmann & MacDonald, 1977).

8.3 Occupational exposure

The evaluation of the effects of alpha-HCH on occupationally
exposed workers is seriously hampered by the fact that most of the
relevant studies concern workers who were exposed during the
manufacture and handling of lindane or the handling and spraying of
technical-grade HCH among other pesticides, and were thus exposed to
all HCH isomers plus impurities and other (process) chemicals.
Therefore, it is difficult, if not impossible, to relate the observed
effects to individual substances. Consequently these studies have only
been described in this monograph where they aid the evaluation.

Behrbohm & Brandt (1959) described 26 cases of allergic and toxic
dermatitis that arose during the manufacture of technical-grade HCH.
Patch testing with pure alpha-, beta-, gamma-, and delta-HCH yielded
negative results, but positive reactions were obtained with the
residual fractions.

The level of alpha-HCH in 57 healthy workers (with normal liver
function, EMG and EEG) at a lindane-manufacturing plant ranged from 10
to 273 µg/litre, whereas it was below the detection limit in control
workers. The concentration in the adipose tissue of eight of the
exposed workers ranged from 1 to 15 mg alpha-HCH/kg (in extractable
lipids) (Baumann et al., 1980, 1981; Brassow et al., 1981; Tomczak et
al., 1981).

The mean serum alpha-HCH level of malaria-control workers that
sprayed technical-grade HCH for 16 weeks increased from 10 to
78 µg/litre in previously non-exposed workers and from 18 to
77 µg/litre in those that had been exposed during three previous
spraying seasons (Gupta et al., 1982).

Nigam et al. (1986) studied 64 employees from a plant
manufacturing HCH who were directly or indirectly associated with the
production of this insecticide and thus also exposed to chemicals such
as benzene and chlorine. The exposed group was composed of 19
"handlers" (who handled and packed the insecticide), 26 "non-handlers"
(plant operators and supervisors exposed indirectly to HCH), and 19
maintenance staff (who visited the plant frequently). The control
group consisted of 14 workers who had no occupational contact with the
insecticide. The exposure period varied up to 30 years. The mean
serum alpha-HCH concentrations in the four groups were 21.1 µg/litre
(controls), 21.8 µg/litre (maintenance staff), 41.2 µg per litre
(non-handlers), and 100 µg/litre (handlers). Lindane and beta- and
delta-HCH were also present. The total HCH concentrations were 51.4,
143.6, 265.6, and 604 µg per litre, respectively. Clinical examination
revealed that the majority of the workers from the "handler" and
"non-handler" groups exhibited paraesthesia of the face and
extremities, headache, and giddiness, and some of them also showed
symptoms of malaise, vomiting, tremors, apprehension, confusion, loss
of sleep, impaired memory, and loss of libido. The same symptoms were
found among the maintenance staff but were less severe and less
frequent.

Chattopadhyay et al. (1988) studied 45 male workers exposed to
HCH during its manufacture and compared them with 22 matched controls.
Exposure was mainly via the skin. Paraesthesia of face and
extremities, headache, giddiness, vomiting, apprehension, and loss of
sleep, as well as some changes in liver function tests, were reported
and were found to be related more to the intensity of exposure (as
measured by the HCH levels in blood serum) than to the duration of
exposure. The measured exposures to total HCH were 13 to 20 times
higher than those in the control groups (no detailed figures were
reported). Of the total serum HCH, 60-80% was beta-HCH.

9. EFFECTS ON OTHER ORGANISMS IN THE LABORATORY AND FIELD

9.1 Algae

Palmer & Maloney (1955) used alpha-HCH in a preliminary screening
test with two cyanobacterium (blue-green alga), two green alga, and
two diatom species. The test concentration was 2 mg/litre of water,
and the incubation period was 3-21 days. Alpha-HCH was not toxic at
this concentration.

When Canton et al. (1975) exposed Chlorella pyrenoidosa to
alpha-HCH for 96 h at 28°C (static system), the EC₅₀ (growth
inhibition) was > 10 mg/litre (maximum solubility in the medium).

In a study by Krishnakumari (1977), cultures of the green alga
Scenedesmus acutus of 1, 3, or 5 days of age were tested for
sensitivity to alpha-HCH at 28°C, the growth rate being used as a
parameter. Alpha-HCH dissolved in ethanol was added at nominal
concentrations of 0.5-100 mg/litre water. The alpha-HCH concentrations
that caused a reduction in growth in 1-, 3-, and 5-day-old cultures
were 10 (or more), 5, and 0.5 mg/kg, respectively.

When Chlamydomonas sp. was exposed at a temperature of 20-25°C
in a static system, the no-observed-effect level (based on the growth
in 48 h) was > 1.4 mg/litre. A similar result was obtained with
Dunaliella sp. at 15°C and a study duration of 48 and 96 h, the
NOEL for growth being 1.4 mg/litre (maximum solubility) (Canton et
al., 1978).

9.2 Protozoa

The EC₅₀ for Tetrahymena pyriformis (3 days in closed system
at 27°C) was reported to be 0.75 mg/litre (Mathur et al., 1984).

9.3 Invertebrates

9.3.1 Acute toxicity

The result of acute or short-term toxicity studies lasting a few
days on Artemia salina, Daphnia magna, and Lymnaea stagnalis are
summarized in Table 3.

Table 3. Acute or short-term toxicity of alpha-hexachlorocyclohexane for invertebrates

Species Age Temperature Parameter Concentration References
(°C) (mg/litre)

Artemia salina 3 weeks 24 LC₅₀^a,b 0.5 Canton et al. (1978)

Daphnia magna < 1 day 20 LC₅₀^c,d 0.8 Canton et al. (1975)

Lymnaea stagnalis 6 months 22 EC₅₀^c,e 1.2 Canton & Slooff (1977)

^a synthetic saltwater
^b 35 days (but exposure time was 4 days)
^c 48 h
^d closed system
^e growth inhibition/mortality or immobilization

9.3.2 Short- and long-term toxicity

9.3.2.1 Crustaceae

In a study by Canton et al. (1975), Daphnia magna was exposed
to 0, 10, 50, 200, 1000, or 2000 µg alpha-HCH (> 95%) per litre for
25 days. The daphnids were fed Chlorella pyrenoidosa. The
sensitivity of daphnids to alpha-HCH markedly increased with exposure
time. A concentration of approximately 50 µg/litre or less did not
lead to death at any time during the whole life cycle of 2 months.
Only with 2000 µg/litre was there an influence on reproduction, the
EC₅₀ for reproduction inhibition being 100 (54-186) µg/litre.
The EC₅₀ based on mortality and immobilization was 800
(600-1000) µg/litre (see Table 4).

9.3.2.2 Molluscs

In a short-term (2-day) study, groups of five adult snails
(Lymnaea stagnalis L.) (6 months of age) were exposed to various
dose levels. Based on mortality and immobility, the EC₅₀ was
estimated to be 1200 (600-2300) µg alpha-HCH (> 95%) per litre
(Canton & Slooff, 1977).

In a long-term (70-day) study, groups of 10 snails (5 months of
age) were exposed to 20, 100, 300, or 600 µg per litre. The study was
divided into a pre-exposure period (14 days) during which all egg
capsules and the number of eggs per capsule were counted, an exposure
period of 40 days during which four groups of adults and five capsules
of each group were exposed to alpha-HCH, and a post-exposure period
(16 days) during which snails were placed in water to recover. Based
on egg production inhibition, the 40-day EC₅₀ was 250 µg/litre. The
percentage of fertilized eggs per capsule was not affected, and no
morphological abnormalities were noticed during embryonic development.
Based on the number of eggs that did not hatch, an EC₅₀ of
230 µg/litre was determined. Considering a combination of the
inhibition of egg production and the mortality of the young during
their development, a 50% reduction of the overall reproductivity was
found at 65 µg alpha-HCH/litre. These effects did not disappear during
the recovery period of 16 days (Canton & Slooff, 1977) (see Table 4).

Table 4. Long-term toxicity of alpha-hexachlorocyclohexane for invertebrates

Species Age Temperature Duration Criteria Concentration References
(°C) (days) (mg/litre)

Daphnia magna 19 21 no mortality; 0.27^a Janssen et al.
no effects on behaviour, 0.09 (1987)
appearance or growth;
no influence on reproduction 0.27
(4 groups of offspring)

Lymnaea stagnalis adults 22 40 EC₅₀ (egg production inhibition) 0.25 Canton & Slooff
eggs and 22 40 hatching, overall productivity 0.065 (1977)
adults

^a water renewal system

9.4 Fish

9.4.1 Acute toxicity

LC₅₀ and EC₅₀ (mortality and immobilization) values for fish
are summarized in Table 5.

9.4.2 Short- and long-term toxicity

During a 3-month study, rainbow trout (Salmo gairdneri)
(200-250 g) were fed pellets containing 0, 10, 50, 250, or 1250 mg
alpha-HCH (purity > 95%) per kg diet. After 2, 4, 8, and 12 weeks,
the fish were examined. Growth, microsomal liver enzymes (aniline
hydroxylase and aminopyrine demethylase), brain cholinesterase, serum
alkaline phosphatase, and the histopathology of the brain, liver, and
kidneys were all investigated but no effects were found (Canton et
al., 1975).

When guppies (Poecilia reticulata) aged 3-4 weeks were exposed
to 0, 200, 800, or 2000 µg alpha-HCH (> 95%) per litre in a 50-day
study, the EC₅₀, based on mortality and immobilization, was 800
(600-1200) µg/litre (Canton et al., 1975).

In a study by Janssen et al. (1987), fertilized eggs of Oryzia
latipes were exposed for 35 days (up to 28 days after hatching) to
alpha-HCH. No influence on growth, mortality or behaviour was seen at
800 µg/litre.