United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Cincinnati OH 45268
EPA-600 ''1-78-047
December 1978
Research and Development
Reviews of the
Environmental
Effects of
Pollutants:
XII.
Hexachlorocyclopentadiene
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document \s available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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.EEA-600/1-78-047
December 1978
REVIEWS OF THE ENVIRONMENTAL EFFECTS OF POLLUTANTS:
XII. HEXACHLOROCYCLOPENTADIENE
by
Mary Anne Bell, Robert A. Ewing and Garson A. Lutz
Battelle
Columbus Laboratories
Columbus, Ohio 43201
Contract No. 68-03-2608
Date Published: October 1979
Project Officer
Jerry F. Stara
Office of Program Operations
Health Effects Research Laboratory
Cincinnati, Ohio 45268
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Health Effects Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products constitute
endorsement or recommendation for use.
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FOREWORD
A vast amount of published material is accumulating as numerous
research investigations are conducted to develop a data base on the adverse
effects of environmental pollution. As this information is amassed, it
becomes continually more critical to focus on pertinent, well-designed studies.
Research data must be summarized and interpreted in order to adequately
evaluate the potential hazards of these substances to ecosystems and
ultimately to public health.
The series of documents entitled "Reviews of the Enviornmental Effects
of Pollutants" (REEPs) represents an extensive compilation of relevant
research and forms an up-to-date compendium of the environmental effect data
on selected pollutants.
The Review of the Environmental Effects of Hexachlorocyclopentadiene
includes information on the chemical and physical properties of both compounds;
pertinent analytical techniques; transport processes to the environment and
subsequent distribution and deposition; impact on microorganisms, plants, and
wildlife; toxicologic data in experimental animals including metabolism,
toxicity, mutagenicity, teratogenicity and carcinogenicity; and an assessment
of their health effects in man.
The REEPS are intended to serve various technical and administrative
personnel within the Agency in the decision-making processes; i.e. in the
development of criteria documents and environmental standards, and for other
regulatory actions.*
* The breadth of these documents makes them a useful resource for
public health personnel, environmental specialists, and control
officers. Upon request these documents will be made available
to any interested individuals or firms, both in and out of the
Government. Depending on the supply, you can obtain the
document directly by writing to:
U.S. EPA
Environmental Criteria and Assessment Office
26 W. St. Clair Street
Cincinnati, Ohio 45268
Jerry F. Stara
Director
Environmental Criteria and
Assessment Office
111
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PREFACE
Hexachlorocyclopentadiene ("hex") is one of those organic chemicals
manufactured and used in substantial quantities within the chemical
industry, but which are almost unknown outside the industry. Although it
is an important intermediate in the manufacture of a number of
organochlorine pesticides and flame retardants, it has essentially no end
uses of its own. Thus, it is not surprising that its potential impact
upon the environment has been studied very little, and that little is
known about its environmental behavior, or its effects upon the biosphere
or upon humans.
This KEEP document is an attempt to gather together and recapitulate
what is known about hex. It is painfully clear that there are large gaps
in the information on its effects, particularly at chronic or sub-acute
levels. More needs to be learned about hex to ensure that its use in
manufacturing pesticides and flame retardants useful to man is safely
conducted, so that environmental problems do not arise.
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ABSTRACT
The objective of this research program was to assemble in a publicly
- available document information on the environmental effects of
hexachlorocyclopentadiene (hex), a topic on which the published
literature is still extremely sparse. A significant fraction of the
information contained in this review consists of heretofore unpublished
information made available by the two U.S. manufacturers of hex.
Hex was used in the past in large quantities for the production of
numerous important organochlorine pesticides, including chlordane,
aldrin, dieldrin, heptachlor, and endrin, as well as such minor,, but
well-known pesticides as mirex and Kepone. At present, only Pentac and
endosulfan are produced in significant quantities. The other current
major use of hex is in the manufacture of flame retardants for plastics
and polymers, a large and still growing market.
Since hex is basically a chemical intermediate with essentially no
end uses of its own, hex concentrations in the environment should be
negligible, and limited data suggest that this indeed is the case.
Probably contributing to this is its ready disappearance through
hydrolysis and photolysis. Due to its infrequency in the environment and
its low profile as an intermediate, there have been few studies of the
behavior of hex in the environment or in biological systems. Data on
chronic exposures are especially lacking.
Very little is known regarding potential hex exposures to the general
public through ingestion of contaminated food or water- Hex has been
detected in waters near points of industrial discharge and in a few
samples of indigenous fish, but elsewhere there appears to be almost no
information on concentrations in surface waters, or in foods. The
heaviest and most chronic exposures to hex undoubtedly occur among
persons engaged directly in the manufacture of hex and among production
workers fabricating hex-containing products. Inhalation is the primary
mode of occupational exposure.
Extremely limited data are available concerning the effects of hex
exposure on humans. A recent incident in which sewage treatment plant
workers were exposed accidentally provides some information on acute
responses, however, no systematic epidemologic studies of chronically
exposed individuals have been reported.
Animal studies have demonstrated that hex is quite toxic via oral,
dermal, and inhalation exposures. Chronic inhalation experiemnts have
shown that inhalation of less than 1 ppm hex produced fatalities as well
as a variety of pathological changes in several species of animals. To
vii
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date, satisfactory subchronic and chronic oral toxicity studies have not
been completed. Several attempted studies have failed to establish an
oral dose which could be tolerated without mortality over an extended
period of time. Similarly, little is known about the metabolism of hex.
It appears that the compound is readily absorbed by the lungs, skin and
stomach tissues and fairly rapid excretion occurs through the urine,
feces and possibly through the respiratory tract. Standard toxicological
tests for mutagenicity and teratogenicity have reported negative results ,
however once again, the extreme toxicity of the compound restricted the
dosages used in these tests to extremely low concentrations of hex. These
tests suggest that outright toxicity, rather than chronic effects, is
perhaps the critical effect of hex, even at very low doses. Evaluation of
the potential carcinogenicity of hex has not been possible due to the
absence of chronic animal studies and epidemiologic studies.
This report was submitted in fulfillment of Contract NO. 68-03-2608
by Battelle's Columbus Laboratories under the sponsorship of the U.S.
Environmental Protection Agency. This report covers the period from
September 21, 1977 to September 30, 1978, and work was completed as of
October 31, 1978.
viii
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CONTENTS
Foreword iii
Preface v
Abstract vii
Figures xi
Tables xi
Acknowledgements xiii
1.0 General Summary/Environmental Assessment 1
1.1 Technology of Hexachlorocyclopentadiene 1
1.2 Hexachlorocyclopentadiene and the Environment 3
1.3 Animal Toxicity 4
1.4 Human Toxicity 6
2.0 Technology of Hexachlorocyclopentadiene 8
2.1 Characterization of Hexachlorocyclopentadiene 8
2.1.1 Physical Properties 8
2.1.2 Chemical Properties 9
2.1.3 Stability 10
2.1.4 Analysis 10
2.1.4.1 Commercial Hexachlorocyclopentadiene . . 10
2.1.4.2 Environmental Samples 11
2.2 Methods of Preparation 13
2.1.1 Laboratory Preparation 13
2.2.2 Commercial Manufacture 13
2.3 Uses of Hexachlorocyclopentadiene 16
2.3.1 Pesticides. . . ft 16
2.3.1.1 Thiodan 16
2.3.2.2 Pentac 18
2.3.2 Flame Retardants 19
2.3.3 Miscellaneous Uses 25
2.4 Production of Hexachlorocyclopentadiene 27
3.0 Hexachlorocyclopentadiene and the Environment 32
IX
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CONTENTS (Continued)
3.1 Effects on Microorganisms 32
3.2 Effects on Aquatic Biota 33
3.2.1 Insects 33
3.2.2 Fish 33
3.3 Effects on Terrestrial Biota 34
3.4 Transport and Fate 35
4.0 Animal Toxicity 39
4.1 Acute and Subacute Tests 39
4.1.1 Oral Administration 39
4.1.2 Cutaneous Administration 39
4.1.3 Inhalation Tests 41
4.2 Chronic Toxicity 42
4.2.1 Oral 42
4.2.2 Dermal 44
4.2.3 Inhalation 45
4.3 Symptoms and Pathological Effects 45
4.3.1 Oral Administration 45
4.3.2 Dermal Application 4?
4.3.3 Inhalation Tests 4?
4.4 Comparative Toxocity 49
4.5 Metabolism 49
4.6 Teratogenicity 52
4.7 Mutagenicity 53
4.8 Carcinogenicity 57
5.0 Human Toxicity 61
5.1 Detection Thresholds 61
5.2 Louisville Contamination Incident 63
5.2.1 Plant Employee Health Effects Evaluation 65
5.2.2 Comunity Survey 70
5.3 Carcinogenicity 70
6.0 References 71
x
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FIGURES
2.1 Synthesis of the diene group of chlorinated pesticides from
hexachlorocyclopentadiene 17
5.1 Employees who noticed unusual odor at plant, by day,
Louisville, Kentucky, March 1-28, 1977 66
TABLES
2.1 Summary of Analysis of Technical Hexachlorocyclopentadiene . . 12
2.2 Description and Properties of Chlorendic Anhydride
and Chlorendic Acid 21
2.3 Potential Applications of Hex and Chlorendic Acid
Type Flame Retardants 22
2.4 Available Hex-Based Flame Retardants 23
2.5 Smoke from Various Halogenated Resins 26
2.6 Production/Sales of Pesticides Manufractured from
Hexachlorocyclopentadiene 29
2.7 Flame Retardant Use in Plastics 30
4.1 Dose Response Data: Inhalation of Hex Vapors 43
4.2 Pilot Teratology Study in Rats: Caesarian Section Data
for Individual Females 54
4.3 Summary of Mouse Lymphoma Results 56
5.1 Analysis of Sludge Sample from Louisville, Kentucky
Wastewater Treatment Plant 64
5.2 Symptoms of 145 Plant Employees, Louisville, Kentucky,
March, 1977 68
5.3 Attack Rates in Employees, By Main Work Area 69
5.4 Case Attack Rates in 124 Employees Exposed to
Plant Work Areas 69
XI
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ACKNOWLEDGEMENT
The cooperation of the Velsicol Chemical Corporation and of the
Hooker Chemicals and Plastics Corporation in making available for this
study pertinent information on hexachlorocyclopentadiene from unpublished
internal company investigations and reports is gratefully acknowledged.
We are particularly indebted to Dr. Whitacre, Director of Environmental
Sciences of Velsicol and Dr. Mitchell Zavon, Medical Director of Hooker,
for their perception in recognizing and responding to the need for a
wider dissemination of these basic data on hexachlorocyclopentadiene to
the scientific community.
We also wish to express our appreciation for the cooperation
annd support received from the EPA staff on the Health Effects Research
Laboratory during the preparation of this document, particularly to Dr.
Jerry F. Stara, the project officer, who provided needed support and
encouragement throughout the program. He was ably assisted by Bonita M.
Smith and Karen L. Blackburn. The support of Dr. John R. Garner, Director
of HERL, was much appreciated.
XI11
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1.0 GENERAL SUMMARY/ENVIRONMENTAL ASSESSMENT
Hexachlorocyclopentadiene, hereafter referred to as hex, is a highly
reactive, highly chlorinated compound, which is the key intermediate in
the manufacture of many important organochlorine pesticides and flame
retardants for organic polymers. As such its production has been
significant, touching 22.5 million kg (50 million pounds) per year at
times.
Hex is an important chemical intermediate, but it has essentially no
end uses of its own. Thus, hex concentrations in the environment should
be negligible, and in general this is indeed the case. Small
concentrations of hex were occasionally present as impurities in
pesticides made from it, and some entered the environment this way.
However, the most likely route for its entry into the environment arises
from the manufacturing process, of either hex or products made from hex.
These in fact represent the only documented sources of hex in the
environment.
Because of its infrequency in the environment, and the low profile it
maintained as a chemical intermediate, there have been but few studies of
its behavior and data are exceedingly limited; most of the data contained
in this environmental review represent laboratory investigations.
However, by the same token, hex has heretofore not presented a major
environmental problem, except in isolated instances such as the disposal
of hex manufacturing wastes to the Louisville, Kentucky, sewer system.
This introductory chapter will provide a general summary of the more
detailed coverage provided in the subsequent chapters. As indicated
above, the sparseness of the available data precludes a very detailed
treatment. Most of the experimental studies which have been conducted on
hex have been performed by the Hooker Chemicals and Plastics Corporation
and the Velsicol Chemical Corporation, the only two U.S. producers of
hex. Without the data which these two firms have made available, this
review of the environmental effects of hex would have been much more
limited.
In the interest of organization, the topics will be reviewed and
commented on in the order in which they appear in the document.
1.1 TECHNOLOGY OF HEXACHLOROCYCLOPENTADIENE
Hex, C Cl,, is a pale yellow nonflammable liquid having a very
pungent odor, soluble in a number of organic solvents but with a very low
solubility, ca. 2 ppm, in water- Its boiling point is 239 C (H62F) and it
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is sufficiently volatile at ambient temperatures to have a tendency to
disperse to the atmosphere. This tendency to volatilize may also explain
why its presence is found in so few environmental samples.
Its two double bonds make it a highly reactive compound which readily
undergoes substitution and addition reactions. Its versatility is based
upon its reactivity as a diene with a variety of olefins and polynuclear
aromatic hydrocarbons in the Diels-Alder reaction. It is a key ingredient
in the production of the cyclodiene group of chlorinated pesticides,
including a number which had a large commercial market, e.g. chlordarie,
aldrin, dieldrin, heptachlor, isodrin, endrin, mirex, and Kepone .
Environmental considerations have led to the banning, suspension, or
severe restriction of the use of these pesticides. Only two hex-based
pesticides have escaped these restrictions, and are still freely .used,
endosulfan (Thiodan ), a broad spectrum insecticide, and Pentac an
acaricide used primarily for the control of mites in greenhouse
cultivation.
Hex, unlike some of the pesticides derived from it, degrades rapidly
by photolysis, giving water soluble degradation products. Tests on its
stability towards hydrolysis at ambient temperature indicated a half-life
of about 11 days at pH 3-6, which was reduced to 6 days at pH 9. In an
aqueous solution, hex can disappear after as little as 30 minutes of
photolysis.
Producers specifications for commercial technical hex call for 97.5
percent minimum hex content; principal contaminating chlorine compounds
are hexachlorobutadiene (C^Cl,), and octachlorocyclopentene (C Clfi). Of
the impurities present in commercial hex, hexachlorobutadiene is Ihe most
important, since it goes through a Diels-Alder reaction unchanged and may
carry through to the product; its toxicity is indicated to be greater
than that of hex.
Commercial hex is purified by distillation, with higher-boiling
compounds such as hexachlorobenzene (CgCl,-) and octachlorocyclopentene
reporting in the distillation residue. Disposition of these by-products
can create some environmental problems unless properly handled;
octachlorocyclopentene appears to be less toxic than hex on the basis of
some rather limited data.
Hex has only two major types of uses. Its use in the manufacture of
organochlorine pesticides has already been mentioned. Because of the
restrictions which have been placed upon the major members of this
family, this use has decreased significantly from that of earlier years.
How much is difficult to state quantitatively, since production or sales
data are unavailable, but the main insecticides mentioned were each
estimated to consume 2,000 to 5,000 metric tons/year 10 to 15 years ago,
and chlordane was two or three times this. Thus, current use for
pesticides must be only a fraction of this.
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The principal current use of hex is in the manufacture of flame
retardant compounds for incorporation into plastics and polymers to
confer flame retardant properties on them. The major derivative used for
flame retardants appears to be chlorendic anhydride and chlorendic acid,
made by reacting equimolar quantities of hex and maleic anhydride. Hex
can be reacted with numerous dienes to form other flame retardants ,
generally containing from 55 to 70 percent chlorine.
Beyond these two main uses of hex are only very minor uses, consuming
but insignificant quantities of hex.
Estimates of current annual production rates for hex are uncertain
and variable, as low as 7,200 tons to as high as 25,000 tons per year.
The higher figure may be suspect since one of the two producers is not
currently manufacturing hex.
1.2 HEXACHLOROCYCLOPENTADIENE AND THE ENVIRONMENT
Very little is known about the behavior of hex in natural aquatic and
terrestrial ecosystems. In particular, information on the fate and
transport of this compound is very sketchy. To date only one study has
addressed these issues. Laboratory studies of microorganisms, insects,
and fish have quantified several aspects of acute toxicity of hex using a
rather wide range of test organisms. Unfortunately, there are
insufficient data at present for assessment of the potential for chronic
effects and its persistence in the environment.
Hex is extremely effective as a bacteriocide; 10 ppm hex was twice as
effective in killing the common sewage bacteria Salmonella typhosa and
fecal coliform than equivalent concentrations of chlorine. At the same
concentration, hex appears to exhibit little toxicity to soil microbes,
however. Tests for mutagenic activity (e.g., the Ames Mutagenic Assay)
using several strains of Salmonella typhimurium indicated that although
hex had a repressive (toxic) effect on the test organisms, it was not
judged to be mutagenic.
Static bioassays showed a high level of acute toxicity to Daphnia in
that 1 ppm hex was lethal to 50 percent of the organisms. Similarly, an
LC,-n of only 2.3 ppm was reported for mosquito larvae.
Based on current data, it is difficult to draw any general
conclusions concerning the dosage required to produce acute toxicity to
fish. An examination of the results of various bioassays shows large
discrepancies in LC values reported for aquatic species. For example,
trout and bluegill appeared to be able to tolerate much higher
concentrations of hex than fathead minnows or fathead minnow larvae.
Median tolerance for the trout and bluegill were reported to be 20-35
ppm, whereas the LC for fathead minnow larvae was less than 10 ppb.
Such differences in sensitivity may relate in part to interspecies
differences, life stage of the test organism, characteristics of the test
(e.g., static or flow through systems) and water conditions such as
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hardness and temperature, or more likely some combination of these
factors. Further replication of these tests will be necessary before
recommendations can be made concerning safe concentrations of hex in
natural waters. Rather preliminary evidence suggests that hex may
accumulate in fish tissues, but this has yet to be demonstrated. Model
ecosystem studies indicate the potential for biomagnification through the
food chain, however, the probability of occurrence in the natural
environment cannot be adequately evaluated, since essential information
on the quantities of hex reaching aquatic ecosystems and the behavior of
the compound under natural conditions is lacking.
Even less is known about the effects of hex in the terrestrial
environment. It appears that soil conditions such as moisture and organic
content play a decisive role in determining toxicity to insects. Studies
utilizing radiolabelled hex applied to soil showed substantial losses of
recoverable C-hex; much of this loss was presumably due to
volatilization.
1.3 ANIMAL TOXICITY
The classic studies of hex toxicity to mammals were conducted in the
mid-1950's by Treon, et al. This series of investigations reported on
both acute and subacute toxicity of hex to various species of mammals
under a variety of exposure regimens. Oral, dermal, and inhalation modes
of exposure were included in Treon's experiments. More recent proprietary
studies of the oral and dermal toxicity have become available. In
general, these findings agree remarkably well with those of Treon.
Toxicologic mammal studies of hex subsequent to the 1950's could not be
located in the open literature, probably due to the rather low profile of
the compound relative to other pesticide chemicals.
Oral LD-Q values for hex lie in the range of 420-620 mg/kg in rabbits
and about 500 mg/kg for rats. Thus, in terms of acute toxicity to rats,
hex is intermediate between Kepone (LD 95-140 mg/kg) and mirex (LD -
365-740 mg/kg), two closely related pesticides. In contrast to Kepone ana
mirex, hex is nearly as toxic via dermal application as it is following
oral administration.
Hex also is an extremely potent irritant and accordingly has been
classified as an "extreme irritant and corrosive substance" based on eye
irritation tests as specified by FDA under the standards set by the
Federal Hazardous Substances Act.
Inhalation tests indicate that hex vapors were extremely toxic to all
four species of animals (guinea pigs, rats, mice, rabbits) employed in
Treon, et al's tests. In fact, these tests reported that hex was more
toxic than either phosgene or carbon tetrachloride. Despite some
interspecies differences in sensitivity, inhalation of relatively low
levels of hex (1.5-3.2 ppm) was fatal to half of the test animals
following a seven hour exposure. Subchronic tests exposed mice, rats,
guinea pigs, and rabbits to 0.34 ppm in air for 7 hours per day, 5 days
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per week. None of the rats or mice survived more than 20 such exposure
periods. High mortality rates were observed at this dosage among rabbits
as well. Yet another portion of Treon, et al's studies attempted to
examine chronic oral toxicity. Rats and rabbits given various dosages
ranging between 180-2000 mg/kg were fatal within such a short period that
the investigators were unable to establish an oral dosage which could be
tolerated without mortality over an extended period. A Soviet study
reported that oral administration as little as 20 mg/kg for 6 months was
fatal to 20 percent of white rats.
Pathologic examination of the hex-treated animals revealed similar
types of abnormalities following oral, dermal and inhalation modes of
exposure. Diffuse degenerative changes in the brain, heart, liver, kidney
and adrenal glands were characteristic; the extent of damage varied
directly with dosage and duration of exposure. Prolonged intermittent
exposure to as litle as 0,15 ppm of hex vapor induced slight degenerative
changes in the liver and kidneys of all species of animals tested.
Only two studies addressing the pharmokinetics of hex could be
located. One of the studies exposed rats to various doses by gavage; a
second portion of the same study examined guinea pigs exposed to varying
doses of hex via dermal application. Hex was readily absorbed through the
stomach tissues and through the skin. At necropsy both species of animals
showed pathologic and histopathologic findings suggestive of excretion
through the lungs. The second study examined absorption, metabolism, and
excretion of hex following a single dose of radiolabelled hex. Assay of
urine and feces of rats confirmed the presence of the labelled compound
in both media. Furthermore, at least four metabolites were discovered in
the urine although the exact identity of these metabolites is unknown at
the time of this writing.
The same study revealed that urine represents an important route of
excretion of hex. Seven days after administration, approximately
one-third of the total administered dose had been excreted in the rats
urine. Likewise, fecal excretion accounted for as much as 10 percent of
the original dose. The body organs themselves retained only minute traces
of the C-hex, suggesting that less than 1/2 of the total dose could be
accounted for but must have been eliminated by routes other than the
urine or feces. Thus, this study also provides indirect evidence that
excretion through the respiratory tract may be the primary mode of
elimination of hex. Obviously, such a suggestion must be regarded as
tentative pending confirmation from further studies.
Tests for teratogenicity (a pilot teratology study in rats) and
mutagenicity (a dominant lethal study and several short term iri vitro
tests) have been reported as negative, however, it should be noted that
extremely low doses were employed due to the toxicity of hex to the test
organisms. Thus, the predictive validity of the results at higher dosages
is uncertain.
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Presently available data do not permit evaluation of the carcinogenic
potential of hex. The required chronic animal studies have not been
conducted, nor have any epidemiologic studies reported on cancer or on
any other chronic effects in humans.
Due to its suspect chemical structure (based on the
structural-activity relationships mentioned above), its widespread use an
an intermediate in many cyclodiene pesticides and the relative lack of
data on the effects of chronic exposure, hex has been selected for
testing as part of the National Cancer Institute's test program.
Hoepfully, further testing will clarify both the carcinogenicity issue
and provide needed data on the chronic effects of hex on one or more
mammalian species.
1.4 HUMAN TOXICITY
Extremely limited data are available concerning the effects of hex
exposure on humans; no systematic epidemiologic studies of individuals
chronically exposed to this compound have been reported to date.
Consequently, most of what can be said about human health effects is
based on inferences from animal studies and a few isolated incidents of
accidental human exposure. Potential modes of human exposure are
uncertain at this time. In particular, it is unknown whether oral
exposure (e.g., through ingestion of hex-contaminated drinking water)
constitutes a significant source of human exposure. Dermal and inhalation
exposures undoubtedly occur among workers directly engaged in hex
manufacture and probably among those engaged in the formulation of other
related pesticides where hex may be present as an impurity. Recently, a
group of sewage treatment plant workers were exposed to acutely toxic
levels of hex arising from the clandestine disposal of large quantities
of the compound to the Louisville, Kentucky, municipal sewer system.
Based on the findings of animal studies in which prolonged
intermittent exposure of animals to hex concentrations as low as 0.15 ppm
induced slight degenerative changes, prudence would dictate strict
limitation of human exposure. Persons having opportunities for skin and
respiratory contact with hex should be equipped with, and trained in the
use of appropriate protective clothing and respiratory protection. The
present Threshold Limit Value (TLV) for industrial exposure is set at
0.01 ppm or about 7 percent of the lowest vapor concentration shown to
produce chronic toxic effects in laboratory animals.
Most people are capable of detecting the presence of hex in air at
concentrations as low as 0.33 ppm by its pungent odor. Experimental
studies have shown that some individuals could detect as little as 0.15
ppm. Laboratory workers developed headaches following incidental exposure
to relatively low concentrations of hex vapor present in respiratory
chambers used in animal experiments even after evacuation of the hex
contaminated air and flushing with clean air. Although air samples were
not taken during these episodes, a reasonable estimate would place the
concentration which elicited the headaches in the 0.15 to 1.0 ppm range.
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A single documented incident of acute human exposure occurred at the
Morris Forman Wastewater Treatment Plant in Louisville, Kentucky. The
problem apparently began in March, 1977, when an unknown odoriferous
sticky material began entering the plant and gummed up equipment in the
Screen and Grit Building of the plant. Workers attempting to remove the
sticky material by steam cleaning experienced severe irritation of the
eyes, nose, throat, lungs, and skin. Several of the men required medical
treatment for these symptoms, but none were hospitalized.
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2.0 TECHNOLOGY OF HEXACHLOROCYCLOPENTADIENE
2.1 CHARACTERIZATION OF HEX
2.1.1 Physical Properties
Hex is a pale yellow nonflammable liquid having a very pungent odor.
It is soluble in all proportions in acetone, carbon tetrachloride,
ethanol, and hexane at 25 C. (Forette, 1977). The solubility of hex in
water is 2.10 ppm at 25 C and 2.25 ppm at 35 C (Dal Monte and Yu, 1977).
The vapor pressure, as determined by Lanksmen (1978), can be represented
by the equation:
Log VP = (-3050.
1-9.03638
T K
and is 0.063 mm at 25 C.
Typical data and product specifications drawn from Hooker (1964) and
Velsicol (1975) product bulletins are:
Grade:
Specifications:
Synthesis
Other Properties:
Assay Hex, Minimum
Chlorine Content
Tetrachlorocyclopentadiene
Octachlorocyclopentene
Specific Gravity, 15.5/15.5
Free Chlorine, ppm maximum
HC1, ppm maximum
Iron, ppm maximum
Molecular Weight
Boiling Point, 760 mm
Boiling Point, 10 mm
Melting Point
Freezing Point Depression
Viscosity, cps 25 C
Flash Point (Open Cup)
Lb/gal at 15.5 C
Refractive Index n.
25
Surface Tension
Latent Heat of Fusion
97.5
78
0.6-0.8 wt.
1.7-1.9 wt.
C 1.700-1.715
2.5
20
3
273
239 C
108 C
9.5-9.9 C
16.1 C/mol
7.8
None
14.2
1.5625 +.001
47 dynes/cm
2712 cal/gm
percent
percent
percent
percent
mol
Fairly extensive investigations of the spectra of hex, its
derivatives, and bicyclic structures containing the same moiety have been
8
-------
reported. (Idol, et al., 1955; McBee, et al., 1955; Ungnade and McBee,
1958; and Brooks, 1974).
Hex has an absorption band in the ultraviolet at 322 m and 323 m
(log e = 3.17) in ethanol. Allylic chlorines replaced by hydrogens shift
the maximum absorption to lower wavelengths but increase the molar
absorptivity. The infrared spectrum of the diene has two bands in the
double-bond stretching region at 6.24 and 6.3 nm (1603 and 1572 cm ),
6.225 and 6.35 nm (1606 and 1575 cm" ), and three bands in the C.-C
stretching region at 12.45, 14.21, and 14.75 nm (803, 704, and 678 cm" ).
The Raman spectrum gives two bands in the double-bond region at 1606 and
1572 cm" (Ungnade and McBee, 1958).
2.1.2 Chemical Properties
The two double bonds make hex a highly reactive compound which
undergoes substitution and addition reactions to provide a variety of
intermediates, for example, acids, acid halides and anhydrides, esters,
amides, ketones, diketones, quinones, nitriles, and other halogenated
hydrocarbons. The versatility of hex is based upon its reactivity as a
diene with a variety of olefins and polynuclear aromatic hydrocarbons
(Whetstone, 1964, and Roberts 1958) in the Diels-Alder reaction. These
products are generally 1:1 adducts containing a
hexachloro(2 .2. 1 )bicycloheptene structure. If the dieneophile contains
multiple unsaturation, hex may form both mono-adducts and di-or
poly-adducts . Most of the insecticides are mono-adducts. The flame
retardant products are both mono and diadducts (Rosenberg, 1978)
Diels-Alder additions with hex are generally batch reactions in
stirred glass-lined kettles at 50-150 C for 5 to 20 hours. Olefin is
usually in excess and the reaction normally occurs at atmospheric
pressure unless the olefin is low boiling. The products are usually
isolated as bottom products after vacuum stripping or are crystallized.
The reaction of hex with maleic anhydride leads to chlorendic
anhydride and by hydrolysis to chlorendic acid, a key intermediate in the
manufacture of flame-resistant resins.
Although steric isomers are possible in the condensation of hex with
olefin, the products invariably contain the substituent derived from the
olefin in the endo position. Products with the substituents in the exo
configuration, are formed in small amount, if at all (Whetstone, 1964).
With polycyclic olefins, products of a more complicated nature are
formed, for example, exo-endo, exo-exo-, endo-endo, and endo-exo
stereoisomers. Hex adds reversibly to polynuclear aromatics such as
naphthalene and anthracene to yield potential intermediates in the
manufacture of dyes (Look, 1974). Reduction of hex by catalytic
hydrogenation in the presence of platinum or palladium catalysts proceeds
stepwise to give pentachlorocyclopentadiene, tetrachlorocyclopentadiene
and finally cyclopentane (McBee and Smith, 1955). Dechlorination of hex
-------
with zinc dust and ethanol yields cyclopentadiene. Both
hexachlorocyclopentadiene and its reduction products are decomposed by
alkaline substances (Brooks, 1974).
2.1.3 Stability of Hexachlorocyclopentadiene
A number of studies on the stability of hex under various conditions
have been conducted in the laboratories of the Velsicol Chemical
Corporation.
14
The photolysis of an aqueous solution of C hex with light from a
mercury-vapor lamp (medium pressure mercury vapor) was investigated by Yu
and Atallah (1977a). Of the total energy radiated from the lamp about 40
to 48 percent was in the ultraviolet portion of the spectrum, 40 to 43
percent in the visible, and the remainder in the infrared. Gas liquid
chromatography of the petroleum ether extract from the photolysis showed
that the hex degraded rapidly to water soluble products. No hex was
detected in the aqueous solution following 30 minutes of photolysis.
Studies on the hydrolysis of hex at various pH's and temperatures
were also conducted by Yu and Atallah (1977b). C hex was used to study
the stability of hex at pH 3,6,9, and 12 at 25 C. At pH 12 the half-life
of hex was less than 2 hours. At 25 C, the half-lives were 9.2, 10.6 and
4.4 days, at pH 3,6, and 9, respectively. Thin layer chromatography (TLC)
was used to separate the hex and its degradation products. Gas liquid
chromatography was employed to confirm the C hex spots from thin layer
chromatography.
As noted above, the boiling point of hex is 239 C (462 F), and the
vapor pressure at 25 C is only 0.063 mm Hg. On this basis hex would be
characterized as a relatively non-volatile substance, and would not be
expected to be particularly labile. However, field evidence indicates
that this is not the case, and that it disappears rapidly from aqueous
and terrestrial substrates. This is evidently true even in the
laboratory. Whitacre (1978) reports that even hex standards in organic
solvents will degrade under laboratory conditions. It is common practice
in Velsicol laboratories to cover volumetric flasks containing hex
solutions with aluminum foil and keep them refrigerated while not in use.
Otherwise, losses of 50 percent of hex can occur within a few days.
2.1.4 Analysis
2.1.4.1 Commercial Hexachlorocyclopentadiene —
As noted earlier, commercial technical hex may contain a number of
other chlorinated hydrocarbons. Wysocki and Rozek (1977) assayed a
"typical" production lot sample of technical hex for tetrachloroethylene
(TCE), hex (C-56), hexachlorobutadiene (C-46), hex ketone,
octachlorocyclopentene (C-58), h exa ch 1 orobenzene (HCB),
pentachlorobenzene (CgCl H) and mirex. Analysis was done by gas
chromatography and component identifications confirmed by mass
10
-------
spectrometry. Summarized results are shown in Table 2.1. Hex manufactured
by other procedures may contain slightly smaller or larger concentrations
of contaminants.
2.1.4.2 Environmental Samples --
Analytical methods for hexachlorocyclopentadiene are similar to those
used for other chlorocarbons. Gas liquid chromatography (GC) is the
method of choice, generally using an electron-capture detector. The
Pesticides Analytical Manual, Volume I; Methods Which Detect Multiple
Residues (U.S. Department of Health, Education, and Welfare, 1977) gives
detailed descriptions of methodology, instrumentation, interfering
substances, and confirmatory tests for chlorinated pesticides, much of
which is applicable to the analysis of hex. Retention time is the
principal parameter used to discriminate between compounds with GC
analysis. This is a non-specific characteristic, and accurate analyses
with GC depend critically on careful techniques and rigid control of
operating parameters.
Some of the potential difficulties in the analysis of samples
containing hex have been identified by Eichler (1978):
(1 ) Previously developed analytical methods may not be applicable to the
specific problem in question, e.g. new interferences may be present
(2) Hex adsorbs rapidly onto metal surfaces in a gas chromatograph. Thus,
it is good practice to use only glass coated lines. It also absorbs
to a lesser degree on glass surfaces so that the amount of glass
wool plug in the end of the chromatographic column may affect the
analysis
(3) The location from which the biological sample came must be considered
since the analytical apparatus of choice may not be applicable
(4) In trace levels analysis, contaminants may cause an erroneous
response which may be interpreted as the presence of the contaminant
of interest
(5) Conscientiously avoided must be such well known analytical
difficulties as glassware contamination, solvent impurity, and
variations in solvent purity, sample storage, and instrumental
difficulties such as reproducibility in injection technique
(6) Advances in quantitative analysis have pushed the detectability
limits for organics down to the ppb or ppt level. This has
complicated the analytical scheme because more impurities and
contaminants are now detectable. At present the state-of-the-art for
confirming the structure of this multiplicity of trace contaminants
is gas chromatography/mass spectrometry.
11
-------
TABLE 2.1 SUMMARY OF ANALYSIS OF TECHNICAL
HEXACHLOROCYCLOPENTADIENEa
Component
C2C14
C4C16 (C-46)
Hex (C-56)
C6C15H
C5Clg (C-58)
C6C16
Internal Standard
Mi rex
Typical Retention Time (Min)
1.2
6.3
8.5
11.5
12.8
14.1
21.7
33.5
Average Percent
0.09
1.11
98.25
0.02
0.68
0.04
—
ND°
100.2
Total mass
balance
Source: Wysocki and Rozek (1977).
On basis of GC areas, normalized to hex.
"Not detected
12
-------
2.2 METHODS OF PREPARATION
2.2.1 Laboratory Preparation
Hexachlorocyclopentadiene, C^Cl, (1,2,3,4,5,5-Hexachlorocyclopenta-
diene) has been prepared by several methods which are described in the
review article by Ungnade and McBee (1958). These include:
(1) The chlorination of cyclopentadiene with 6 to 11 mol of aqueous
0.25-4.5 molar sodium or potassium hypochlorite at temperatures
ranging from -5 C to +50 C. Solvents such as benzene, carbon
tetrachloride, chloroform, hexane or isopropyl ether may be
used in up to 10:1 ratio with the cyclopentadiene, but the
reaction may equally well be conducted without solvent. Side
reactions can be minimized by the addition of 1 mol percent
(based on the cyclopentadiene) of sodium sulfamate (Kleinman,
1953) or an emulsifying agent such as sodium lauryl sulfate
(Lidov, et al., 1952). After about 20 minutes of reaction, the
organic layer is separated and fractionated j.n vacuo to give a
55 percent yield of hex, boiling point 60 to 62 C/1 mm. The
main by-product of this synthesis is 1,2,3,4,5
pentachlorocyclopentadiene which on standing gives a dimer,
melting point 214 C. Compounds such as
1,4,5,5-tetrachlorocyclopentadiene can also be isolated from
the low boiling material preceding hex.
(2) The condensation of hexachloropropene and dichloroethylene with
aluminum chloride (Prins, 1937; and Prins et al., 1946).
(3) Stepwise condensation of trichloroethylene with chloroform
followed by dehydrochlorination with aluminum chloride. The
isolated intermediates in the reaction are hexachloropropane
and pentachloropropylene (Prins, 1946).
(4) The dechlorination of octachlorocyclopentene by heat and
catalysts or with hydrogen and platinum. Maude and Rosenberg,
(1956) obtained about 90 per yield of hex when vapors of
octachlorocyclopentene were contacted with a catalytic surface
selected from the group consisting of nickel, cobalt, nickel
chloride, cobalt chloride, and mixtures thereof in a reaction
maintained at about 400-550 C. McBee, et al., (1955) obtained a
49 percent yield of hex from the hydrogenation of a mixture of
octachlorocyclopentene, alcohol and platinum catalyst.
2.2.2 Commercial Manufacture
Rosenberg (1978) in his appraisal of past and current commercially
attractive methods for manufacturing hex, provides information on the
status of raw materials and recoverable by-products and distillation
residues. The following comments can be made, based on his assessment of
the above three parameters.
13
-------
The choice of the starting material depends on a combination of
technical and economic factors. The first report on the preparation of
hex was published by Straus, et al., (1930). In their process,
cyclopentadiene is reacted with an alkaline hypochlorite solution to give
a. crude hexachlorocyclopentadiene which also contains some dimeric
by-products. The hex can be recovered by fractional distillation under
reduced pressure. This procedure was used by Velsicol in developing their
initial group of hex-based insecticides,, and the crude material was
reportedly used to make technical Chlordane .
Exhaustive liquid phase chlorination of a cyclic C-5 hydrocarbon will
provide octachlorocyclopentene (C-58) as the chlorination end product.
This compound can also be made in low yield by liquid phase chlorination
of various aliphatic C-5 and C-6 starting materials.
Newcomer (1953) described the conditions for making hex from
trichlorethylene (C_HC1 ) and carbon tetrachloride (CCl^). Krynitsky and
Bost (1974) reported the thermal vapor-phase dechlorination of
octachlorocyclopentene (C-58) at a temperature of 470-480 C to yield hex.
A procedure employing the preparation of C-58 by liquid phase
chlorination of a cyclic C-5 hydrocarbon followed by vapor-phase
dechlorination of C-58 to hex was developed subsequently and used
commercially by Shell Development Company and is apparently the basis of
the process used in their plant at Pernis in the Netherlands.
Various C-5 hydrocarbons can be used to prepare hex by a variety of
vapor-phase chlorination processes. Because of the large heat of
chlorination, it is usual to prepare a partially chlorinated material by
a liquid phase reaction to be used as a feed to the vapor-phase reactor.
If the parent hydrocarbon is normal or isopentane, a catalyst is needed
in the vapor-phase reaction to obtain a high yield. No hex is obtained
from neopentane. This process was developed by Hooker and operated
successfully for many years. The crude product is a mixture of fully
chlorinated materials. The by-products are mainly chlorocarbons formed by
chain-rupture, such as carbon tetrachloride (CCl^), tetrachloroethylene
(C_C1^), and hexachlorobutadiene (C^Cl,). Hexachlorobenzene (C,C1,-) may
also be present. In the absence of a catalyst, these perchlorinated.
by-products may amount to as much as 50 percent by weight of the crude
product. With the proper catalyst, the amount may vary from 5-15 percent
of the crude product.
With the increasing availability of dicyclopentadiene from the
thermal cracking of petroleum fractions in the late sixties, the use of
this alternate raw material became attractive. This material is readily
cracked to form cyclopentadiene which can be chlorinated in the liquid
phase to obtain tetrachlorocyclopentane or more highly chlorinated
cyclopentanes. Subsequent thermal or catalytic vapor-phase chlorination
at elevated temperatures produces hex. Under closely controlled
conditions no significant by-products are formed (except for the
co-produced C-58). Under severe conditions various side reactions are
-------
observed. It is postulated that transient C_C1 free radicals are
produced, as evidenced by the by-products which are formed. These free
radicals may either trimerize to form C,C1, , dimerize and chlorinate to
form C|.Clg, or chlorinate to form C Cl.. The concentration of these
by-products increases at high reaction temperatures.
In summary, in commercial operation crude hex may thus contain the
arying proportions: CClj., C Cl^, C
chlorocarbons are present, sinc
o fragments to form CClj. and C Clj..
l hydrogen may also be present, bu
following chlorocarbons in varying proportions: CClj., C Cl^, ^,,
C Cl , C Clg , C,C1,. No C-3 chlorocarbons are present, since the
percnlonnated compound C_Clo fragments to form CClj. and C Clj.. Trace
compounds containing residual hydrogen may also be present, but the
amount is usually negligible.
Fractional distillation of crude hex usually results in virtually
complete separation of CCl^, C?C1^, and C.-C1 , . The distilled product will
contain varying amounts of C^CI, and C Ci depending on the composition
of the crude product and the efficiency of the distillation.
Specifications for the commercial product permit small amounts of these
by-products. For most commercial uses, no further treatment of the
distilled product is needed. The CClj., C Cl^, and C^Cl, are recovered as
an overhead fraction in the distillation system. If a cyclic hydrocarbon
is used as the starting material, the amount is too small to have
commercial value. The C,C1, together with some CCCIQ is removed as a
molten distillation residue. Pure C&C1, is a nigh-melting (229 C)
material which has low solubility in tne other chlorocarbons in the
crude. The residue is usually solid or semi-solid at room temperature.
Of the impurities present in commercial hex, the most important is
hexachlorobutadiene (C-46). Most hex derivatives are made by a
Diels-Alder condensation reaction. Any C-46 present is unchanged in the
process and may carry through to the product, although, since most
Diels-Alder condensations are carried out in a solvent, the unreacted
C-46 is removed with the solvent. Published data on the toxicity of
hexachlorobutadiene indicate that it is more toxic than hex. Commercial
products such as aldrin may contain as much as 3 percent C-46. The C-46
content will depend on the commercial source of hex. The commercial
product made by Hooker since installation of the dicyclopentadiene
process contains very little 0-46.
McBee, et al. , (1950 and 1953), and Maude and Rosenberg, (1953 and
1956) described a two-stage process for the preparation of hex from
pentane, isopentane, or mixed pentanes. The hydrocarbon mixture is
chlorinated photochemically in the liquid phase at 80 to 90 C until an
average composition of C,-H Cl with a density of 1.63 to 1.70 is
obtained. The subsequently vaporized chloropentanes are passed in a vapor
of chlorine over a Floridin catalyst maintained at 350 to 400 C followed
by passage through an upacked section of tubing heated at 500 C. By
controlling the thermal chlorination in this manner, octachloropentene is
produced which at 500 C gives a better than 90 percent yield of hex.
15
-------
2.3 USES OF HEXACHLOROCYCLOPENTADIENE
The major uses of hex have been in the manufacture of chlorinated
organic pesticides, initially, and more recently in the manufacture of
flame retardants for polymeric materials. Other miscellaneous uses have
been relatively insignificant.
2.3.1 Pesticides
Hex has been the chemical intermediate used for the production of
numerous chlorinated pesticides, several of which have enjoyed very large
usage. The list includes chlordane, aldrin, d.ieldrin, heptaghlor,
isodrin, endrin, mirex, Kepone, endosulfan (Thiodan ), and Pentac . The
routes of synthesis of these and other pesticides from hex are shown
schematically in Figure 2.1. With the exception of endosulfan and Pentac,
both of which are in current use, the usage of hex-based pesticides has
been banned, suspended, or severely restricted by governmental action.
Although yields in all reactions are good, they are not quantitative.
There is reason to suspect that in some cases free hex may have been
present in the marketed pesticide products. For example, technical
dieldrin is an insecticidal product containing not less than 85 percent
of dieldrin and not more than 15 percent of insecticidally-related active
compounds; technical heptachlor consists of 72 percent actual heptachlor
and 28 percent related compounds; chlordane contains about 60 percent of
the isomeric alpha - and beta - chlordanes and 40 percent of
insecticidally related compounds — chlordene, heptachlor, and nonachloro
analogs; technical endrin contains 95 percent of endrin (Whetstone, 1964).
2.3.1.1 ThiodanR—
P
Endosulfan, (Thiodan ) CgHgClgO S; 6,7,8,9,10,10-Hexachloro-1,5,
5a,9,9a-hexahydro-6,9-methane-Z,4,3^benzodioxathiepin-3-oxide (I) is
manufactured by the reaction of hex with 1,4-diacetoxy-2-butene, followed
by hydrolysis of the diacetoxy derivative to the diol, and reaction of
the diol with thionyl chloride (Frensch, 1957, as quoted by Brooks,
1974). The diol intermediate can also be made directly by the Diels-Alder
condensation of butenediol with hex, the procedure used by Hooker
(Rosenberg, 1978b).
Endosulfan is a broad spectrum insecticide useful for the control of
pests of deciduous fruits, vegetables, and ornamentals, especially of
aphids, leaf hoppers, and spittle bugs. (Hooker, 1975).
The technical material is a brownish solid, melting range 70-100 C,
insoluble in water, stable toward dilute mineral acids and hydrolyzed
rapidly by alkalies. It has an oral LD to the rat of 110 mg/kg.
Technical endosulfan consists of about 4 parts of alpha-isomer, m.p.
108-190 C, and one part of beta-trans isomer, m.p., 206-208 C. The
alpha-isomer, which is a somewhat more potent insecticide, is slowly
converted to the more stable beta form at high temperatures. Both isomers
16
-------
ISODRIN
ENORIN
DIELDDIN
AICI3, S:O2 OR FULLERS
EARTH IN CCI< OR Cf,H6
OR SO2CI2< BENZOYL
PEROXIDE IN C6H6
ENDOSULFAN
EPOXIDATION
H2O2 OR PER ACIDS
H2
C5-HYDROCAR8ONS
DICYCLOPENTA
DIENt
.SO
KfPONE
- Ptrehbrlnolfd Ring
FIGURE 2.1. Synthesis of the diene group of chlorinated pesticides from hexachlorocyclopentadiene.
Source: Lawless, Von Rumker, and Ferguson (1972).
-------
are slowly oxidized in air and in biological systems and rapidly by
peroxides or permanganates to endosulfan sulfate, m.p. 181-182 C (Brooks,
1974). Endosulfan hydrolyzes to the corresponding diol and sulfur dioxide
and when heated under reflux with methanolic sodium hydroxide, gives
sodium sulfite which can be titrated iodometrically. The sulfur dioxide
may be detected colorimetrically (red color) with Rosaniline and
formaldehyde and the absorbance at 570 nm (Brooks 1974).
Another colorimetric analytical method, which involves the treatment
of endosulfan with pyridine and methanolic KOH has been applied in
residue analyses to the determination of both isomers in hexane extracts
with cleanup.
The easy conversion of endosulfan into the parent diol, which can be
detected by GC methods as the diacetate or bis-trimethylsityl ether, has
been used for derivitization analysis of the compound in residues
(Cochrane and Chau, 1971). Beta-endosulfan in the solid state exists in
one modification with a highly symmetrical crystal form (I) and another
with low symmetry (II). For (I) the SO band In the infrared spectrum is
at 1,192 cm and for (II) it is at 1,180 cnf (Maier-Bode, 1968).
Gorbach, et al., (1971) investigated the environmental stability of
Thiodan in the water and soil of treated rice fields. Biotests with
native fish and chemical analyses were carried out to determine the
biological effectiveness of residues in submerged paddy fields in the
vicinity of Pandaan (East Java) during the end of the rainy season in
March, 1970. Thiodan residues declined rapidly, within 3 to 5 days, in
the treated test rice fields. Terminal residues in the water amounted to
0.5-0.0 ppb. Fish were able to tolerate short time exposure to endosulfan
concentrations 4 times the LC . In the mud of submerged as well as
dried rice fields, only low residue concentrations (1.9 ppm maximum) were
found. The increasing sulfate equivalent in the total residue pointed to
decomposition of the pesticide. After appropriate extraction all samples
in this work were analyzed by gas chromatography.
Studies in dogs, rats, mice and flies suggest that when endosulfan is
ingested the sulfate appears briefly in the tissues, especially fat, and
may appear in the milk of animals producing it, but such residues
disappear rapidly when exposure ceases. The half-life of excreted
products^n the urine and feces of sheep given a single dosage of 14
mg/kg of C-endosulfan was about 2 days and the radioisotope level in
milk fell to negligible proportions within 4 days. (Brooks, 1974).
2.3.1.2 Pentac —
p
Pentac , C10ciin> is tne trademark of Hooker Chemicals and Plastics
Corporation (T96tf) for the acaracide bis-(pentach1oro -
2,4-cyclopentadien-1-y-l). It is prepared by the reductive dechlorination
of hex (Ungnade and McBee, 1958; Ladd, 1956; and Rucker, 1960). A 73
percent yield is obtained by coupling two molecules of hex in 80 percent
ethanol or methanol at ambient temperature with cuprous chloride or
18
-------
powdered copper bronze in light petroleum solvent (b.p. 100 C) or by
refluxing with copper powder in toluene (Ungnade, and McBee, 1958).
Reduction of hex with hydrogen at atmospheric pressure using a palladium
on carbon catalyst gives about a 20 percent yield of Pentac (Brooks,
1974).
Pentac is a tan crystalline solid, m.p., 122-123 C, b.p. (decomposes
at 250 C), vapor pressure 10 mm mercury at 25 C. It is insoluble in hot
alcohol, aliphatic hydrocarbons, and moderately soluble in aromatic
hydrocarbons. Pentac is stable towards aqueous acids and bases and can be
safely stored for extended periods. (Hooker, 1968; Martin and Worthing,
1971!). Hookers Pentac WP formulation contains 50 percent active
ingredients. Pentac is recommended for mite control of greenhouse floral
plants and nursery stocks, including roses, chrysanthemums,gardenias,
carnations, azaleas, delphiniums, snapdragons, zinnias, and poinsettias.
It is also effective on outdoor roses and nursery arbor vitae, including
hemlock and spruce. Pentac apears to act by an interference with
ovipositing of eggs by the female mites; initial results require 3 to 5
days. Application is at the rate of 8 ounces per 100 gallons of water
which is sufficient to spray 2,000 to 3,000 mature bushes. Two
applications should be made about 2 weeks apart. It has no insecticidal
activity and is nonphytotoxic. (Allen, et al., 1964; Hooker, 1968).
The acute oral LD of Pentac (technical material and the
emuslsifiable concentrate) for male albino rats is in excess of 3160
mg/kg; the dermal LD Q for albino rats also is greater than 3160 mg/kg.
There was no evidence or systemic toxicity which could be attributed to
percutaneous absorption. A single application of 3-0 mg of Pentac to
rabbit eyes produces a slight irritation which subsided by the 6th day.
(Hooker, 1968).
When heated at 130 C for several hours or when exposed to UV light or
sunlight Pentac suffers pronounced loss of its acaricidal activity
(Brooks, 1974). Pentac is analyzed by infrared spectroscopy with
absorbance at 7-98 run (Hooker, 1968; Martin and Worthing, 1974).
2.3.2 Flame Retardants
The increasing public emphasis on safety, accompanied by an expanding
array of government regulations, has provided the impetus for a large and
growing market for hex-derived chlorinated organic flame retardants.
Flame retardant chemicals enable a material to resist burning when
exposed to a relatively low-energy ignition source, such as a match,
candle, or cigarette.
Hex-derived chlorinated organic compounds are used as flame
retardants primarily in plastics, including polypropylene, polyethylene,
nylon, thermosetting resin, rigid polyurethane foams, unsaturated
polyesters, and other polymers, including epoxy resins (Sanders, 1978).
Rough estimates of 1976 consumption of major alkyd coating resin
reactants indicate that chlorendic anhydride finds limited use in
19
-------
fire-resistant paints for military applications (Chemical Economics
Handbook, 1977). Annual consumption probably does not exceed 250-300 tons.
These additives have the advantage of withstanding relatively high
processing temperatures, but generally have to be used at high loading
levels.
Examination of the chemical technology concerning flame retardants
suggests that beginning about 1958 hex and some of its derivatives,
notably chlorendic acid and some of its derivatives, began to become
increasingly important in this field (Pattison and Hendersinn, 1971).
Chlorendic anhydride (C H 0 Cl,) can be prepared by heating equimolar
amounts of hex and maleic anhyaride in chlorobenzene at 140 to 150 C for
8 to 10 hours (Pattison and Hendersinn, 1971). Water is then added to the
hot anhydride solution to effect hydrolysis and convert the anhydride to
chlorendic acid monohydrate with better than 90 percent yield (after
washing with chlorobenzene and water). Drying at 100 to 105 C yields an
essentially anhydrous product of 99 percent purity:
CL ,C1
Cl
CI
0
chlorendic
anhydride
H,0
C1
chlorendic
acid
Properties of the anhydride and acid are listed in Table 2.2. Table 2.3
lists the wide variety of polymer and coatings systems in which use of
hex and chlorendic acid type flame retardants is recommended.
Hilado (197*0 lists Hooker Chemicals and Plastics Corporation and
Velsicol Chemical Corporation as manufacturers of a number of flame
retardants, (Table 2.4), which are believed to be hex-based. Some
clarification is needed concerning the present role of hex and C..-C1 ?
products manufactured by Hooker for fire-retardant applications. Although
Dechlorane 510 and 4070 (mirex) are shown as products, these were
discontinued on October 1, 1972 (Rosenberg, 19?8b) . Among products
available from Hooker, the 1977 Buyer's Guide Issue of Chemical Week
lists Dechlorane Plus 25, Dechlorane Plus 515 (chlorine content 65
percent) and Dechlorane 602 (chlorine content 69 percent). However,
production of Dechlorane 602 and 604 has been discontinued (Rosenberg,
1978b).
"Dechlorane is a trademark of Hooker Chemicals and Plastics Corporation
20
-------
TABLE 2.2. DESCRIPTION AND PROPERTIES OF CHLORENDIC ANHYDRIDE
AND CHLORENDIC ACID3
Molecular weight
Percent chlorine
Appearance
Chlorendic
Anhydride
370.85
57.4
White crystalline
Chlorendic
Acid
388.87
54.7
White chrystalline
Melting point
Volatility
solid
240 - 241 C
Very low at 25 C
solid
Decomposes to chlorendic
anhydride
Very low at 25 C
Solubility (25 C)
lg/100 g solvent)
Benzene
Hexane
Acetone
Carbon tetrachloride
Linseed oil (raw)
Water
40.4
4.5
127.0
6.7
19.3
Hydrolyzes to chlorendic
acid
1.1
0.1
144.0
0.4
9.4
0.4C
Source: Adapted from Velsicol Commercial Development Technical Bulletin
No. 524 (1961 ).
3Chlorendic anhydride sublimes at 90-100 C at a pressure of 0.5 mm mercury.
"Ca 7.0 at 94 C.
21
-------
TABLE 2.3. POTENTIAL APPLICATIONS OF HEX AND CHLORENDIC
ACID TYPE FLAME RETARDANTS&
Flame Retardant Recommended
Dechlorane Plus
515 & 25
Dechlorane 602b
Dechlorane 603
Dibutyl chlorendate
Dimethyl chlorendate
Chlorendic acid
Chlorendic anhydride
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of Modern Plastics Magazine. (c) McGraw
Reprinted by permission
Hill, Inc. (1977).
Production has been discontinued.
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22
-------
TABLE 2.4. HEX-BASED FLAME-RETARDANTS'
Chlorine, Percent
Hooker Chemical Corporation
Dechlorane plus 25
Dechlorane plus 515
Dechlorane 510 (mirex)
Dechlorane 4070 (mirex)
Dechlorane 602b
Dechlorane 603
Dechlorane 604
C-56
HET acid
HET anhydride
(hexachlorocyclopentadiene)
(chlorendic acid)
(chlorendic anhydride
Velsicol Chemical Corporation
Douse 499
Dimethyl chlorendate
Dibutyl chlorendate
Hex-cod (hexachlorocyclopentadiene-cyclooctadiene)
65.1
65.1
78.0
78.0
69.4
66.7
78.0
54.7
57.4
42.7 (32.0)'
51.1
42.5
55.0
aSource: Adapted from Hilado. Reprinted with permission from Flammability
Handbook for Plastics. (C) Technomic Publishing Company, Inc. (1974)
Has been withdrawn from the market.
°Did not achieve commercial status.
Also contains 32.0 percent bromine.
23
-------
The halogen content of some of the current flame retardants that may
represent products of more recent technology suggests that hex continues
to be a key ingredient in their manufacture. For example, Pattison and
Hendersinn (1971) report that bichlorendo (one of the earlier C1(,C112
products manufactured by Hooker) has been replaced in many applications
by 1,5-bis (chlorendo) cyclooctane. This compound is prepared by a
Diels-Alder reaction of 2 equivalents of hex and 1,5-cyclooctadiene as
indicated in the equation below:
Cl
(5)
l,5-bis{chlorendo) cyclooctane
Bis (chlorendo) cyclooctane is used extensively with antimony oxide in
flame retardant polypropylene and ABS (acrylonitrile-butadiene-styrene)
formulations. Velsicol's Hex-Cod is probably obtained from the reaction
of one equivalent of hex with 1,5-cyclooctadiene since the product
contains 55 percent chlorine. Velsicol also manufactures Cytex B-56, a
1:1 adduct with 1,5 cyclooctadiene, followed by the additions of one mole
of bromine to the remaining double bond.
Employing equivalents of diolefin and one or two equivalents of hex,
the calculated chlorine content of the adducts resulting from each of
several formulations is as follows:
Formulation Product Chlorine, Percent
2 moles hex
1 mole cyclooctadiene 65.1
1 mole hex
1 mole cyclooctadiene 55.8
2 mole hex
1 mole butadiene 70.9
1 mole hex
1 mole butadiene 65.1
2 mole hex
1 mole cyclopentadiene 69.5
1 mole hex
-------
1 mole cyclopentadiene 62.8
2 mole hex
1 mole vinylcyclohexene 65.1
1 mole hex
1 mole vinylcyclohexene 55.8
Also, the reaction of 1 mole hex with 1 mole cyclopentadiene followed
by the addition of 1 mole of bromine would be expected to give a product
containing 32 percent bromine and 42.7 percent chlorine. These
compositions correspond to the halogen contents of Velsicol's Douse 499.
Thus, there seems little doubt that hex continues to be a key chemical in
the preparation of flame retardants for plastics. In most cases,
hydrocarbons have been modified with one or two equivalents of hex.
Beyond this, chlorendic acid and the corresponding anhydride also are
used in preparing flame-retardant resins and plastics.
Miller et al., (1976) investigated smoke and toxic gas emission from
burning unsaturated polyesters (Table 2.5). Concerning chlorendic acid
resin systems they observed that the smoke contained a high level of
hydrogen halide ( 125 ppm per g of resin) but contained considerably
less carbon monoxide than smoke from various halogenated resins.
2.3-3 Miscellaneous Uses
As discussed above, the current major uses of hex are found in ±he
manufactureRof pesticides, at present primarily endosulfan (Thiodan ),
and Pentac , and flame retardants for resins and plastics. There are many
hex and chlorendic acid derivatives described in the patent and technical
literature for which production and consumption figures are not
available. Some of the recommended uses for these derivatives include
bactericides, fungicides, plant growth regulators, weed eradicators,
extreme pressure lubricants, rust inhibitors, flame-resistant composites
with wood, rot-resistant additives in plywood, adhesives for rubber and
plastics, and catalyst activators.
There is believed to only one registration ("Perma-Trim") for the use
of hex as a contact herbicide; in this application it is recommended for
use as a 0.5 percent solution along the edges of walks and driveways,
where total eradication is desired. This use is quite minor, less than a
ton per year.
In comparison with the markets for hex in pesticides and flame
retardants, the consumption of hex at present for these minor uses is
believed to be quite small, and in some cases can be only of
informational and not technological interest. Examples of miscellaneous
publications and patents concerning hex and hex-based derivatives are:
Enhancement of Flame-Resistance of Wool. Friedman, M. , R. E. Whitfield
and S. Tillin, 1973. Textile Research Journal 43; 212-217. Made wool
-------
TABLE 2.5. SMOKE FROM VARIOUS HALOGENATED RESINS3
Resin System
Atlac 711-054
( t etrab romob isphenol-A)
Hetron 92
(chlorendic acid)
Smoke ,
D max/g.
177
93
HX,C
ppm/g
24
>125
CO,
ppm/g
144
70
CoRezyn 925
(tetrachlorophthalic
anhydride)d 124 >55 85
Dion 6125
(dibromohydrophthalic
anhydride)6 94 15 168
Tetrabromophthalic anhydride
(lab cook)e
FR-1540 124 6 139
(dibromoneopentyl glycol)
plus EG resin6 115 1.0 143
aSourc.e: Miller, et al. Reprinted by permission of Modern Plastics
Magazzne. (C) McGraw-Hill, lnc. (1976).
Atlac, ICI America, Inc.; CoRezyn, Interplastic Corp.; Dion, Diamond
Shamrock Chemical Co.; FR-1540, Dow Chemical; Hetron, Hooker Chemical
Corp.
CHX, X = Halogen
dProbably rated Class II by ASTM E84.
eProbably rated Class I by ASTM E84.
26
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fabrics effectively flame-resistant by reaction with chlorendic anhydride
in dimethylformamide.
Dyebath Application of Chlordenic Acid for Flame-Resistant Wool. 1974;
Friedman, M., J. F. Ash, and W. Fong, Textile Research Journal 44;
555-556. Satisfactory flame resistance was obtained with chlorendic acid
concentrations of about 8 percent.
Antiwear Formulation, Hammond, J. L., Conte, A. A., Jr-, 1976. Wear
36(3); 387-90. Two weight percent dibutyl chlorendate included in a
poly-(chlorophenyl)methyl siloxane fluid.
Hydraulic Fluid, Page, W. C. and Holbrook, G. W., Dow Corning Corporation
Belgian Patent 839,860 1076. Six weight percent dibutyl chlorendate added
to a liquid siloxane for usage in brakes and steering.
Solvent Extraction of Copper-, Berger, S. A., 1976; Talanta 23(6),
475-479. The use of chlorendic acid was investigated as a function of pH
in the extraction of cupric copper.
Dielectric Fluid, Brooks, W. T. , 1976. Dow Corning Corporation, German
Offen 2,608,447. To a liquid polyorganosiloxane was added 0.5 to 2.0
weight percent chlorendic acid or its esters containing C.-C.~ alkyl
groups. Improved corona initiation voltage and corona extinction voltage
were noted.
Accelerated Curing and Testing of Copolymer Finishes on Wood Panelings,
Paszner, L., R. Szymani, and M. M. Micko, Holzforsch Holzverwert 27, 4-5.
A mixture of tetraethylene glycol dimethylacrylate and chlorendic
anhydride gave the highest hardness and chemical and abrasion resistance
but had poor ultraviolet resistance.
Polymeric Photopolymerization Initiators, Wagner, H. M., J. S. Foster,
Lowman, R. C., 1975. Research Disclosure 134, pp 19-21. Chlorendic
anhydride was used in the preparation of a photopolymerizable ester.
Extreme Pressure Additives for Lubricating Oils, Fields, E. K., A. Steiz,
Jr., Standard Oil Indiana. 1977. U. S. Patent 4,025,569.
2.4 PRODUCTION OF HEXACHLOROCYCLOPENTADIENE
Hex has been manufactured in the United States only by the Velsicol
Chemical Corporation at Memphis, Tennessee, and by the Hooker Chemicals
and Plastics Corporation, initially at Niagara Falls, New York, and in
recent years at Montague, Michigan. In 1977 Hooker discontinued hex
manufacture at Montague, making Velsicol's Memphis plant the only U.S.
producer.
Although Hooker and Velsicol were the only U.S. producers of hex, the
Shell Chemical Company was a major user of hex in the manufacture of
organochlorine insecticides (aldrin, dieldrin, and endrin) at its Denver
27
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plant at Genk, Belgium, also produces chlorendic acid from hex
(Rosenberg, 19?8b). No information was identified on either imports or
exports of hex or hex derivatives; neither is believed to have been a
major factor in U.S. statistics.
Production statistics on hex are unavailable, and estimates are
fairly broad, partly because production has risen and fallen with the
usage of its major insecticide products. Pesticide production/sales
estimates for 1962 and 1972 (Table 2.6) provide an indication of U.S.
consumption of hex for these purposes.
Whetstone (1964) stated that the annual production of hex in 1962 for
the preparation of cyclodiene insecticides must have been at least 22.5
million kg (50 million Ib). Lu, et al., (1975) also concluded that the
production in 1972 for the same purpose could not have been less than 50
million pounds. The above estimates of hex production for the preparation
of cyclodiene insectides seem quite realistic for the period in question.
Because of the restrictions which have been placed on the major
chlorinated insecticides current production for these uses appears to be
only a small fraction of these levels.
The other major use for hex was in the manufacture of
flame-retardants. In 1968, approximately 7,725 metric tons (17 million
pounds) of reactive flame retardants were used for unsaturated
polyesters, of which about 5,000 metric tons (11 million pounds) were
chlorendic acid. As shown in Table 2.7 the current consumption of flame
retardants approximates 175,000 metric tons, of which chlorinated
paraffins and chlorinated cycloaliphatics (the latter would include hex),
chlorendic acid, and anhydride, and derivatives of both hex and
chlorendic acid command a sizeable market. Chlorendic anhydride is the
largest volume reactive chlorinated flame retardant used (Levek and
Williams, 1976).
As reported in Chemical Economics Handbook (1976) approximately 3
million pounds of raaleic anhydride were used in 1974 for the production
of about 10 million pounds of chlorendic acid and anhydride. Since
equimolar quantities of hex are used, and its molecular weight is about
2.77 times that of maleic anhydride, production of this quantity would
require about 8.3 million pounds (4,150 tons) of hex.
The same source projects a growth rate of about 10 percent per year
from 1974 through 1980 due to increasing use of flame retardant polyester
resins for corrosion resistant ductwork and building panels, appliance
parts, and insulation for electrical apparatus. This suggests that about
5 million pounds of maleic anhydride (and nearly 14 million pounds of
hex) will be used in the manufacture of 16.6 million pounds of chlorendic
acid and anhydride in 1980.
Aside from the general statement (above) on the use of chlorendic
acid and anhydride in flame retardant polyester resin formulations, very
28
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TABLE 2.6. PRODUCTION/SALES OF PESTICIDES MANUFACTURED
FROM HEXACHLOROCYCLOPENTADIENE
Estimated Annual Total, Metric Tons (S.T.)
Pesticide 1962 &——-
Chlordane
Aldrin
Heptachlor
Endosulfan
Dieldrin
Endrin
Mirex
n
Pentac
2,250-4,500
—
2,250
450-900
2,250-4,500
2,250-4,500
—
—
(2,500-5
(500-1
(2,500-5
(2,500-5
,000)
,000)
,000)
,000)
11,340
4,500
2,700
910
. 450
—
45C
(12,500)
(5,000)
(3,000)
(1,000)
(500)
(50)c
a Whetstone, et al.,(1964).
b Lu, Po-Yung, et al. ,(1975) .
c Estimated current production.
29
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TABLE 2.7 FLAME RETARDANTS USED IN PLASTICS3
Thousands of
Metric Tons,
Type
Additives
Alumina hydrates
Antimony oxide
Boron compounds
Bromine compounds
Chlorinated paraffins and cycloaliphatics
Phosphate esters, nonhalogenated
Phosphate esters, halogenated
Others
Total
1975
60.0
8.5
4.0
9.0
30
18.7
13.5
5.0
148.7
1976
70.0
11.0
4.7
9.5
35
21.0
17.5
6.0
174.7
Source: Adapted from Modern Plastics (1976). Reprinted by permission by
Modern Plastics Magazine. (C) McGraw-Hill, Inc. (1977).
30
-------
little information was found concerning the specific applications of
these raw materials in coatings.
An estimated production figure of 7200 tons per year of hex has been
reported for the 1975-1976 period (U.S. Environmental Protection Agency,
1977). This estimate may be low. Some industry estimates suggests 2.5 to
5 million kg/yr (5 to 10 million Ib) production of both chlorendic acid
and chlorendic anhydride, and total annual hex production of the order of
22 million kg (50 million Ib).
Hex is sold as a distilled liquid of high purity in nonreturnable
55-gal lined drums (700 Ib) and in 8,000 gal. tank cars. Hex is not
extremely corrosive and can be stored and handled in steel without
harming product or container, if moisture is rigorously excluded.
However, to avoid possibility of iron contamination and corrosion, glass,
nickel, or baked phenolic coatings are recommended (Hooker, 1964). It is
classified as a Class B poison under DOT Regulations, and has a poisonous
liquid NOS, Class B Freight Classification (Hooker, 1969).
31
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3.0 HEXACHLOROCYCLOPENTADIENE AND THE ENVIRONMENT
In this section the effects of hex on microorganisms and on some
aquatic and terrestrial biota are discussed. The amount of information is
not large, and because there are very few field data on hex, the
following evaluation is based almost entirely upon laboratory studies.
3.1 EFFECTS ON MICROORGANISMS
Hexachlorocyclopentadiene (hex) has been shown to have bactericidal
properties and is reported to have germicidal activities towards many
fungi (Cole, 1954). A static bioassay study of its effects on sewage
effluent showed that hex is more toxic than chlorine in reducing
bacterial counts (Cole, 1954). Ten ppm of hex reduced the total count
from four million to less than 10,000 in two hours,a 90 percent
reduction, whereas chlorine reduced the count by only 45 percent in the
same interval. Both five and ten ppm hex were equally effective against
coliform and Salmonella typhosa and resulted in a 90 percent reduction in
one hour, while chlorine produced a 50 percent reduction. In contrast,
Rieck (1977b) found no effects of hex at 15 ppm on bacterial populations,
actinomycetes or fungi in a sandy loam soil. Rieck concluded that no
significant deterimental effects on microbe populations would result from
treating soil with high levels of hexachlorocyclopentadiene. The
difference in results between Cole and Rieck may be due to the
volatility, degradability, and adsorption of hex.
The Ames Mutagenic Assay conducted by Industrial Bio-test
Laboratories, Inc., (1977) using four to five strains of Salmonella
typhimurium indicated that hex is not mutagenic. The tests were made with
and without metabolic activation, using concentrations of hex up to 2500
ug/10 ul added to the microbial assay plates. Concentrations greater than
10 ug/10 ul produced a possible cidal effect in all but one of the
strains tested; a possible cidal effect occurred at 2500 ug/10 ul or
greater in the remaining strain in the absence of metabolic activation. A
repressive effect was noted in three strains at concentrations below 10
ug/10 ul in the absence of metabolic activation. Volitilate ( volatile
vapors) of hex was also tested on one strain using the vapor from hex
concentrations of up to 2500 ug/10 ul and exposure times of up to two
hours. Again no mutagenic effects were observed and the repressive effect
was again noted.
32
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3-2 EFFECTS ON AQUATIC BIOTA
3.2.1 Insects
Static bioassays using the waterflea, Daphnia magna, illustrate that
hex can be lethal to daphnids (Union Carbide Environmental Services,
1977). Daphnids are found in nearly all types of freshwater habitats and
are preyed upon by many species of fish; therefore they are an important
food web organism. Acute 48-hour toxicity studies of Daphnia were made
using filtered lake water. The concentration of the toxicant lethal to 50
percent of the test populations (LC^) at 24 hours was determined to be
93.0 ppb (range 78.9-109.6; 95 percent" confidence limits); the 48-hour
was 52.2 (44.8-60.9) ppb. The no-effect concentration was less than
.(J ppb. The LC and no-effect levels may be expected to vary with
different temperatures, species, and water qualities. Lu, et al., (1975)
reported an LC of 2.3 ppm for mosquito (Culex pipiens) larva.
3.2.2 Fish
Davis and Hardcastle (1959) conducted static bioassays of several
hydrocarbon compounds including hex, for possible use as herbicides in
Louisiana. The purpose was to evaluate the effects on common fish of
representative responsiveness. The species selected for testing were
bluegill (Lepomis machorchirus) and large mouth bass (Micropherus
salmodes). A median tolerance limit (TLM) was established in soft water
from Bayou de Siard, a quiet cut-off fed primarily by surface water. Both
species showed obvious distress within 5 to 10 minutes after the
introduction of hex; there was a loss of equilibrium and only slight
response to touch; however, no deaths occurred in 24 hours in
concentrations up to 500 ppm. At 48 hours, the TLM was 30 and 35 ppm for
bluegill and large mouth bass, respectively; at 96 hours, it was 25 and
20 ppm.
In another reported bioassay, it was found that hex was lethal at 6
ppm to trout and bluegill in 15 to 30 minutes and lethal to sea lamprey
in eleven hours. Effects at 1 ppm were roughly the same; at 0.1 ppm no
effects were observed for up to 24 hours (Equitable Environmental Health,
1976). These lethal concentrations for bluegill are much lower than those
reported by Davis and Hardcastle (1959).
In a study using early life stages of fathead minnows, (Pimephales
promelas) in a flow-through bioassay system survival was significantly
decreased at 7.3 ppb and above after 4 days of exposure. Most fish were
killed in 4 days and all in 30 days in hex concentrations of 9.1 ppb.
Growth of experimental and control fish was not significantly different
at hex concentrations below 7.3 ppb. A toxicity curve shows that this
compound is a non-cumulative poison (Spehar, et al., 1977).
The toxicity of hex on fathead minnows (Pimephales promelas) was
tested by the U.S. Department of Health, Education, and Welfare in 1956
(cited in the report prepared for The Hooker Chemicals and Plastics
33
-------
Corporation by Equitable Environmental Health, Inc., 1976). Recorded 24-,
48-, and 96- hour TLM values in hard water (emulsion) were 0.075, 0.059,
and 0.059 ppm, respectively. These reported values are eight times higher
than those found by Spehar, et al., for the same species (see above).
Comparison of the results test results of Spehar et al. with those of
earlier studies shows that all three of the compounds tested
( he x ac h 1 o r o c y c 1 o p e n t a d i en e , h exa c h 1 o r o n o r bornadi ene or
heptachloronorbornene) were more toxic to fathead minnows in Spehar's
(1977) study than to this same species and others exposed to these
compounds for similar time periods. The authors attributed the lower
values obtained in this test to the utilization of intermittent flow
exposure systems and/or the use of more sensitive life stages of
development for testing (Spehar et al., 1977).
In examining the findings of hex toxicity to fish, one finds
discrepancies which at present cannot be explained. Differences in
species tested, life stage of the test animal, the characteristics of the
test (e. g. static vs. flow-through bioassays) , and water conditions are
factors which possibly contribute to the variation in results between
studies. In addition, in many cases, the purity of the compound termed
hex was not reported. Thus, differences in purity of toxicant might also
account for variability in bioassay findings.
Hexachlorocyclopentadiene and other organochlorine compounds have
been identified in aqueous discharges from a chemical plant in Michigan
which manufactures hex. (Swanson, 1976). A 72-hour static bioassay of
varying dilutions of the effluent from a plant outfall indicated that the
LC was 61 percent effluent; a 100 percent effluent killed all fish in
one hour and a 75 percent effluent killed all fish in 72 hours (DeKraker,
1976).
Yap, et al., (1975) studied the biochemical effects of various
cyclodiene insecticides. The study involved the iin vitro inhibition of
fish brain ATPase activity by compounds such as aldrin, endrin, dieldrin,
chlordane, heptachlor, and Kepone, (for all of which hex is a chemical
intermediate). Hex itself, however, was not used in this study. Bluegills
were used as the source of brain tissue. Of the 15 cyclodiene compounds
tested, all except dieldrin, isodrin, endrin, pentachlorophenol, and
mirex inhibited both Mg and Na -K ATPase. Dieldrin, isodrin, endrin,
and pentachlorophenol stimulated Mg ATPase; mirex had little effect on
either Mg or Na -K ATPase (Yap, et al., 1975). Although iri vitro
results cannot be translated into known toxicity values for organisms or
extrapolated to hex this information suggests that hex may inhibit ATPase.
3.3 EFFECTS ON TERRESTRIAL BIOTA
Hexachlorocyclopentadiene has been used as contact herbicide on
grasses and weeds (Cole, 1954) such as along sidewalks driveways, and
fences. It reportedly has unusual fungicidal properties (Cole, 1954b) but
Rieck (1977a) reported little effect on soil fungi (see above).
34
-------
The specific effects of hex in the terrestrial environment remain
largely unknown. Impacts of the compound can vary significantly with
environmental conditions. Precipitation and wind can result in the
transportation of the compound to unintended areas, with the possibility
of high concentrations occurring. Concentrations of the substance can be
reduced by volatilization; the rates of volatilization, in turn may be
affected by temperature, moisture and humidity. Thus, concentrations and
persistence can vary from time to time and place to place.
Soil conditions can have major influences of the toxicity of
hex-related substances, (e.g. heptachlor, diazinon, dieldrin). Harris
(1966,1972a, 1972b) has shown that these insecticides are more effective
in moist soils than in dry soils and more effective in mineral soils than
in soils with high organic content. A direct-contact toxicity test of
technical chlordane and its components, one of which is hex, was
conducted using crickets (Gryllus pennsylvanlcus) and picture-winged
flies (Chaetopsis debilis) (Harris, 1972b). Hexachlorocyclopentadiene
proved to be the least toxic of eight compounds tested (technical
chlordane, gamma-chlordane, alpha-chlordane, nonachlor, heptachlor,
chlordene, 1-hydroxychlordene, and hex), showing no toxicity to either
species at a one percent solution after 48 hours. Lu, et al., (1975)
reported a LD,_n of 565 ug/g for hex for house flies (musca domestica)
following a topical application, a concentration much lower than that
reported by Harris (1972b) for other insects.
3.4 TRANSPORT AND FATE
Hexachlorocyclopentadiene enters the environment primarily through
discharges and emissions from pesticide production facilities; some also
enters the environment through pesticides and compounds of which hex is
present as an impurity, e.g. chlordane (Harris, 1972b). Once in the
environment it may be transported by wind, surface and underground water,
streams, and biota.
In December, 1975, hex was qualitatively indentified as a contaminant
in the discharge of a pesticide production plant in Memphis. Later, (May,
1977) the compound was identified in the air near a Michigan pesticide
production plant and in its aqueous discharge, and in fish tissue from
the receiving stream (Spehar, et al., 1977). Hex has also been reported
to be present in soil and bay sediments in the vicinity of a Virginia
pesticide plant long after production was discontinued (Swanson, 1976).
In addition, improper disposal of large quantities of hex resulted in
widespread contamination of sewer lines and shutdown of the municipal
sewage treatment plant in Louisville, Kentucky, as described in Section
5.2.
Evidence presented by Rieck (197^) in an investigation conducted for
Velsicol, indicated that vapors of C-hex (and degradation products)
envolved from treated soil (Maury silt loam), to the extent of 11, 13,
15, 16, 17, and 20 percent (cumulative) after passage of air over the
35
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samples for 1, 2, 3, 5, 7, and 14 days. One could, therefore, deduce that
there is volatility from treated soil, and that the rate decreases with
time.
An interesting aspect of this test was that a little more than half
of the volatile products were trapped by hexane and a little less than
half by a 1:1 ethanolamine solution, indicating that some polar
metabolites were evolved (which were not extractable from the
ethanolamine by hexane). Sohxlet extraction of the residual soils with
hexane and 80 percent aqueous methanol extracted insignifica^t
percentages of the applied C. A total of 38 percent of the applied C
was unextractable as determined by combustion analysis.
14
In another experiment Rieck (1977c) applied C-radiolabeled hex to
soil under various conditions to determine the rate of degradation. At
the start of the experiment, 93 percent of the applied substance was
extractable. After 7 days less than 10 per cent of the applied C-hex was
extractable; 36 percent was extractable in autoclaved soil. Approximately
6.5 to 9 percent of the original C-hex in the non-autoclaved soils was
in the form of metabolized polar products after seven days and declined
to about 3.5 to 5 percent of the total after 56 days. The polar products
accounted for 3 to 4 percent of the total jin autoclaved soil during the
entire period. During the study the total C-hex recoverable declined
from 9^ percent at the initiation of the study to 60-75 percent at seven
days; 50-60 per cent was recoverable at 56 days. The loss of recoverable
C-hex was attributed to volatilization of the compound.
In this study soil which had be,e.n extracted was combusted, so^that
any residual but unextracted C was measured directly as C0?.
Unextracted C was found in these samples and thus was accounted for as
a "bound" residue. Had it not been accounted for it would probably have
been assumed to have volatilized.
Volatilization during extraction is also thought to be the reason why
residue accumulations of hex have not been found in edible fish and why
accumulation in fathead minnows was not demonstrated in the test by
Spehar et al., (1977).
Lu,et al. , (1975) investigated the environmental distribution of
'hex1, chlordene, heptachlor, and heptachlor epoxide in food chain
organisms in two laboratory model ecosystems and iri viftro by sheep liver
microsomes. In the first type of model ecosystem study C-labeled 'hex'
was added directly to the water at approximately 0.1 ppm and allowed to
pass through a food web of plankton, daphnia (Daphnia magna), mosquito
larva (Culex pipien quinquefasciatus), fish (Gambusia affinis), alga
(Oedogonium cardiacum) and snail (Physa sp.).The transfer and
degradation were observed over a 3-day period ^t 80 F (26.7 C). In the
second type of model ecosystem study 5 mg of C hex was topically
applied from acetone solution to grass (Sorghum vulgare) growing on the
terrestrial portion, simulating an agricultural application of 1.12 kg/ha
(1.0 Ib/ac). The model ecosystem was allowed to run for 33 days; maximum
36
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hex concentration in the water (0.031 ppm) was reached after 14 days. The
plants were consoled by the salt marsh caterpillar larvae (Estigmene
acrea) and the C-labeled products entered the terrestrial portion as
fecal products, leaf frass, etc. The organisms in the aquatic portion
were the same as listed for the model aquatic system and the radiolabeled
products were allowed to pass through the system over a 33-day period at
80 F with a 12-hr diurnal cycle and a 500 ft-candle illumination.
At the conclusion of both sets of experiments the radioactivity in
water was extracted in ethyl ether and in the various organisms in
acetone, and evaluated as total parts per million, and for relative
amounts of degradation products by thin-layer chroma£ography,
autoradiography, and liquid scintillation counting of the C-labeled
spots. The residual activity in the extracted substrates was determined
by total combustion analyses as CO . The identification of the
m.etabolites was confirmed by cocnromatography and gas-liquid
chromatography. Final aqueous hex concentration was 0.00024 ppm.
In the view of Lu, et al, hex showed considerable, environmental
stability, and from 22 to 50 percent of extractable C detected in
alage, snails, mosquito, and fish was present as hex. What were denoted
as ecological magnification (EM) factors, based on the final hex
concentration in the water were given as follows: alga, EM 340, snail, EM
929, mosquito, EM 1634 (The latter two appeared to be reversed in the
text; the tabular data indicate mosquito to be EM 929 and snail EM 1634),
and fish, EM 448.
As has been pointed out by Whitacre (1978), the authors appear to
have departed from more generally accepted terminology in describing the
increases in concentration as "ecological magnification". What appears to
be happening here is bioconcentration, referring to the process whereby
chemical substances enter aquatic organisms through gills or epithelial
tissues directly from water. Bioaccumulation is a broader term referring
to a process which includes bioconcentration but also includes any uptake
from dietary sources. The term biomagnification is generally reserved to
describe the process by which tissue concentrations of bioaccumulated
chemical residues increase as these materials pass up the food chain
through two or more trophic levels.
On the basis of the tissue concentrations presented by Lu, et al,
biomagnifications are all less than five, and in one case less than one,
i.e., a negative magnification. Additionally, although the substantial
volatility of hex was recognized, the reported biocentration (ecological
magnification) factors were based on the ratio of tissue concentrations
to the residual aqueous hex concentrations after a 33 day exposure to 80
F and 500 ft-candles of illumination. As noted in Section 2.1.3, hex is
subject to both photolysis and hydrolysis, so that final residual hex
concentrations are quite probably not representative of the overall
behavior of the system.
37
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As suggested by Whitacre (1978), since^the total C in the system
was originally all hex, residual extractable C, which includes residual
hex plus its degradation products, may better serve as an approximation
of non-volatilized hex. Bioconcentration factors so calculated range
between 25 and 80.
While natural conditions in the environment are much more complex
than those in the model ecosystem, the study illustrates that hex does
have the capacity for biomagnification in aquatic ecosystems. Since hex
has no dispersive uses, the existence of this type of situation should be
rare, however, it could occur perhaps as the result of a spill or a
non-permissible discharge.
38
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4.0 ANIMAL TOXICITY
4.1 ACUTE AND SUBACUTE TESTS
The classic studies on acute and subacute toxicity (31 weeks) were
carried out in the 1950's by Treon, et al., (1955). Acute toxicity of hex
by oral, dermal and inhalation routes of exposure was examined in a
variety of animal species including guinea pigs, mice, rabbits, rats, and
monkeys. In addition, the effects of subacute vapor inhalation (150
7-hour exposures over a seven month period) were also studied. Results of
these tests are summarized below.
4.1.1 Oral Administration
Acute toxicity of hex was determined by Treon et al . , (1955) by
administering various dosages of a 5 percent solution of hex in peanut
oil directly into the stomachs of several groups of rabbits and rats. The
data on rabbits indicate that the lethal oral dose, administered as
described above, lies in the range between 420 and 620 mg/kg of body
weight.
Rats showed variation in minimum lethal dose depending on sex. Male
rats were somewhat more sensitive in that the lethal dose was somewhat
less than 280 mg/kg body weight whereas for females, the dosage causing
death was greater than 280 mg/kg. The LDj-_ for male rats was determined
to be 505 mg/kg with 95 percent confidence limits of 387-623. It should
be noted that very few of the test animals survived longer than a week
after oral administration of hex.
The International Resesarch and Development Corporation (IRDC, 1968)
conducted similar studies of the oral toxicity of hexachloro-
cyclopentadiene and octachlorocyclopentene to male albino rats. Each of
the test compounds was mixed in corn oil and administered to the rats at
dosage levels of 100, 215, 464, 1000, 2150 and 4640 mg/kg of body weight.
Five rats were tested at each of the above dosage levels of each compound.
The dose which was lethal to 50 percent of the rats (LD ) was
determined to be 926 mg/kg for hex which is somewhat higher than that
reported earlier by Treon, et al . , (1955). The LD,_0 value reported for
octachloroyclopentene was 1470 mg/kg, indicating a somewhat lower
toxicity for "octa" than for hex.
4.1.2 Cutaneous Administration
In this series of experiments, 93-3 percent hex was applied to the
intact skin of rabbits using the technique of Draize, et al., described
39
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by Treon, et al., (1955). It was determined that the lethal dosage lies
between 430 and 610 mg/kg body weight. Such a thing is remarkable in that
hex appears to be just as toxic via dermal application as by ingestion.
The effects of sublethal concentrations were investigated in both
rabbits and monkeys. In the case of the former, dosages as low as 250
mg/kg induced extreme irritation, purplish-black discoloration of the
skin and subcutaneous edema. Although the skin lesions healed eventually,
damage to the skin in the area of application persisted for many days and
the damage varied in severity and extent with the amount (dosage) of the
material applied.
A slightly different procedure was employed in the cutaneous
exposures of the monkeys. In this case, a series of hex concentrations
(0.001, 0.01, 0.1, 1.0 and 10.0 percent) dissolved in Ultrasene were
applied to five sites of the abdominal skin. Dosage of each of the
solutions was 0.01 ml. No irritation or other changes were noted.
However, when 0.05 ml of the ten percent solution was applied to the back
of a monkey for three consecutive days, the skin became severely
irritated and necrotic. Subsequent experiments using more concentrated
solutions (20, 40, 60 and 90 percent) were applied in the dosage of 0.05
ml on separate areas of the monkey's backs. At all concentrations, there
was discoloration of the skin, ranging from very light to dark tan as the
concentration increased. The discoloration was followed by swelling,
which varied from slight to severe, again depending on concentration. The
highest concentration caused cracking, oozing and serous discharge from
the treated areas; intermediate concentrations produced hardening and
swelling of the skin.
Among guinea pigs, the application of 0.01, 0.10 and 1.0 percent
solutions caused no alterations of the skin, however, more concentrated
solutions (40, 60 and 90 percent) resulted in discoloration, hardening
and necrosis of the skin at the application site. Based on these tests,
it appears that the threshold concentration at which hex in Ultrasene
induces irritation of the intact skin lies between 10 and 20 percent for
monkeys and between 1.0 and 40 percent for guinea pigs.
More recently, the irritant properties of hex were examined in a
study conducted by the International Research and Development Corporation
(IRDC, 1972). These tests were commissioned by Velsicol Chemical
Corporation in accordance with the regulations of the Federal Hazardous
Substances Act.
IRDC *( 1972) reported the results of an investigation of acute dermal
toxicity of hex to rabbits. Four male and four female New Zealand white
rabbits were used in this test. The hair was removed from the back of
each rabbit with electric clippers. Two male and two female rabbits were
used at each of two dosage levels. The test compound was applied in a
single administration to the back of each rabbit at a dosage of 200 or
2000 mg/kg body weight. The area of application was wrapped with a gauze
bandage and occluded with saran wrap. Twenty-four hours later, the
40
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bandages were removed and the backs were washed with water. The rabbits
were observed for mortality for a period of 14 days.
All of the animals which received 2000 mg/kg dosage died within 24
hours after application of the compound. At the 200 mg/kg dosage, both
male rabbits died but the female rabbits survived although both females
exhibited weight loss over the 14 day period. The male rabbits that died
showed weight loss also. In addition, cachexia, marked dermal irritation
and hypoactivity were observed. Skin at the site of application turned
purple in color within a few hours after hex application. Based on these
results, hex was concluded to be "a highly toxic material by the dermal
route of exposure" in accordance with the criteria established under the
Federal Hazardous Substances Act.
Hex was tested for eye irritancy by instilling 0.1 ml of the "test
compound" (which was presumably undiluted liquid hex) into the eyes of
New Zealand White rabbits (IRDC, 1972). The test material was placed into
the conjunctival sac of the right eye of each rabbit; the left eye served
as an untreated control. Damage to the eye was evaluated by instillition
of sodium fluorescein into the eye, followed by examination of the
corneal surface for evidence of ocular damage under ultraviolet light. A
graded scale was used to quantify the extent and severity of damage. The
eyes of the rabbits were checked for corneal lesions at periodic
intervals (at 1, 24, 48, 72 hours post exposure and at 7, 14, and 21 days
post-exposure). Examinations at 14 and 21 days, however, were precluded
by deaths of all of the rabbits on or before the 9th day of observation
period.
Based on the severity of the ocular lesions produced in the rabbits,
hex was concluded to be "an extreme irritant and probable corrosive
substance" in the five minute test and "an extreme irritant and corrosive
substance" in the 24 hour wash test (IRDC, 1972). These classifications
are set in accordance with standards set under The Federal Hazardous
Substances Act, specifically Part 191, Hazardous Substances Test for Eye
Irritants, Food and Drug Administration.
4.1.3 Inhalation Tests
Treon, et al., (1955) exposed various animal species to vapors formed
by bubbling a stream of air through liquid hex contained in a bubbling
tower. This air was then mixed with clean air to achieve the desired
concentration. The stream of air, conditioned with respect to
temperature, dust content and humidity was then passed into a plywood
exposure chamber in which the test animals were confined. A series of hex
concentrations in the air in the exposure chamber were used; these varied
from 0.0017 to 0.804 mg/1 or 0.15 to 73.6 ppm, respectively. Test species
were guinea pigs, rats, mice, and rabbits.
The authors reported that hex vapors were very toxic to all four
species of animals. Exposure to the concentration of 13-0 ppm (an
intermediate level in this experiment) for 15 minutes produced fatalities
41
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in all species except guinea pigs. Of the 4 species, rabbits appeared to
be the most susceptible. Mice, rats and guinea pigs followed in order of
decreasing susceptibility. Table 4.1 depicts the results of the
inhalation experiments. The values tabulated correspond to the
concentration in ppm which: (1) permitted all animals to survive; (2)
killed some, but not all animals and (3) uniformly lethal conditions.
Animals of the following species died regularly when exposed to hex
vapors at the following concentrations and durations: rabbits - 1.5 ppm
for 7 hours; mice - 1.4 ppm for 2 seven-hour periods; rats - 1.0 ppm for
5 seven-hour periods or 3.2 ppm for 2 seven-hour periods and guinea pigs
- 3.2 ppm for 2 seven-hour periods.
When mice, rats, rabbits and guinea pigs were exposed to 0.34 ppm in
air for seven hours a day for 5 days per week, none of the mice or rats
survived more than 20 such exposures. Two thirds of the rabbits had died
by the end of the 25th period, however the guinea pigs survived through
30 periods. At 0.15 ppm, some animals from all four species survived 150
seven hour exposures over a period of 216 days. Eight percent of the mice
did not survive the prolonged intermittent exposure. Details of these
findings are discussed under the heading "chronic toxicity" in the next
section.
IRDC (1972) also reported the results of acute inhalation experiments
in rats. The test animals were exposed to atmospheric concentration of
approximately 2 and 200 mg/1 of the test compound for 4 hours. Due to the
extremely high dosages employed (176.2 and 17624 ppm, respectively)
little information could be derived from the study. No justification of
the choice of dosages was given. All of the animals receiving the test
compound at either exposure level died within 48 hours. All rats at the
200 mg/1 dosage level died during the four hour exposure period. At the 2
mg/1 atmospheric concentration 1 rat died during the exposure period, 8
were dead within 48 hours and 1 died on the second day of observation.
Signs seen during the exposure period included eye squint, dyspnea,
cyanosis, salivation, lacrimation and nasal discharge. Gross necropsy
showed gray coloration of the skin, severe hemorrhage of the lungs and
hydrothorax among rats exposed to 200 mg/1. Rats exposed to 2 mg/1
revealed congestion of the lungs in all cases.
Based on these results the investigators concluded that hex is a
highly toxic material by the inhalation route of administration.
4.2 CHRONIC TOXICITY
4.2.1 Oral
In Treon, et al's., (1955) study, rabbits and rats given various
dosages of hex ranging from 180-2100 mg/kg tended not to survive long
enough at these dosages to provide acceptable data on chronic oral
toxicity. Individuals which survived and were killed subsequent to
42
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TABLE 4.1. DOSE RESPONSE DATA: INHALATION OF
HEX VAPORS3 FOR VARYING EXPOSURE TIMES
Species of Animal
Guinea pigs ....
Rats
Mice
Rabbits
Fatalities,
Percent
0
50
100
0
50
100
0
40
100
0
67
100
Hex Concentration,
1.0 hr
7.2
13.8
20. Ob
3.1
7.2
20. Ob
1.4
7.2
13.8
1.4
3.1
7.2
3.5 hr
3.1
7.1
12.4
1.4
3.1
7.1
1.4b
3.1e
7.1
6.4
7.1
ppm
7.0 hr
1.5
3.2
6.7
1.5C
3.2d
6.7
1.5e
3.2
7.5
Source: Treon,et al., 1955.
Duration of exposure was 1.25 hr.
"25% of group died.
75% of group died.
"80% of group died.
20% of group died.
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exposure showed degenerative changes in the liver, kidney tubules, brain
and adrenal glands. These effects appeared to be at least partially
reversible in that the severity of these lesions diminished as the
interval of survival lengthened (after cessation of oral hex
administration). Symptoms and pathological changes exhibited in these
animals are more fully described under the topic of effects. In any
event, Treon, et al., did not establish an oral dosage which could be
tolerated (e.g. without mortality) over an extended period of time.
Studies in the Soviet Union reported by Naishstein and Lisovskaya
(1965), appear to provide the only source of information on the effects
of long-term, low-dose exposure to hex. Investigations of three aspects
of hex toxicity were reported: (1) minimum lethal dose and cumulative
effects; (2) dermal toxicity and (3) effects of prolonged ingestion of
low doses of hex. Naishstein and Lisovskaya found the minimum lethal dose
of hex for white rats was 600 mg/kg body weight. Note that this value is
equivalent to the upper range of LDCQ'S (420-620 mg/kg) reported by
Treon, et al., (1955) and by IRDC (530-630 mg/kg). No explanation for
this discrepancy is given, however, it should be noted that no assay of
the material designated C-56 was reported by these investigators. Also,
although it was stated that hex was given in oil solution, the exact
method of administration (e.g. mixing with food, intubation, etc.) could
not be determined from the report. Daily administration of 1/30 of the
minimum lethal dose (20 mg/kg) for 6 months killed only 2 animals out of
10, even though the cumulative dose received was 1.5 times the LD , or
uniformly lethal dose. Although some changes were noted in the weight
coefficients of the internal organs of the animals, the authors judged
the cumulative effects of hex to be weak.
4.2.2 Dermal
Treon, et al., (1955) reported that dosages of less than 10 percent
hex appeared to be tolerated without irritative effects in monkeys and
probably also in guinea pigs. Unfortunately, the authors did not continue
the low dose regimen for a sufficient period to observe chronic effects.
High concentrations, 430-6130 mg/kg, applied to the skin of rabbits were
frequently fatal within a few hours. Six rabbits which survived for 7-21
days after application of hex were killed and autopsied. Degenerative
changes were seen in the brain, liver, kidneys and adrenal glands of
these animals in addition to chronic skin inflammation, acanthosis;
hyperceratosis and epilation. Visceral lesions due to dermal hex
application reported by Treon, et al. , (1955) are described in the
section on pathological effects.
Naishstein and Lisovskaya also investigated the effects of prolonged,
low-dose dermal exposure to hex. These experiments consisted of applying
0.5-0.6 ml of a concentration of 20 ppm hex in aqueous solution to the
shaved skin of rabbits daily for a period of 10 days. No differences were
detected between the skin of the experimental animals and that of the
control animals.
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According to Naishstein and Lisovskaya (1965), papers by Soviet and
other researchers have demonstrated the important part played by the
hypopysis-suprarenal cortex system in nonspecific reactions of the
organism to unfavorable factors. Spynu (1959) in particular, in his
studies on the functional state of this system when affected by chemical
agents of low intensity (including poisonous chemicals in diene
synthesis), noted changes in weight and ascorbic acid content of the
suprarenals. Naishstein and Lisovskaya failed to find significant
differences between exposed and unexposed rabbits with respect to these
parameters.
4.2.3 Inhalation
Guinea pigs exposed to a concentration of 0.34 ppm hex for seven
hours per day, five days a week survived until they reached 30 periods of
exposure (6 weeks). However, rats and mice exposed to this concentration
survived only 5 periods of exposure. Survival of the rabbits was
intermediate; 2/3 had died before the end of the fifth week (25 exposure
periods).
A lower concentration, 0.15 ppm hex, was tolerated by guinea pigs,
rabbits and rats throughout 150 seven-hour periods of exposure extending
over a period of approximately 7 months. Four of five mice died within
this period. The guinea pigs, rabbits and rats grew normally during this
period, however, slight degenerative changes were seen in the livers and
kidneys of these animals. These changes are discussed under the heading,
"Pathological Effects."
At the relatively high concentrations, many of the animals died
during the exposure period whereas with the lower exposure levels most of
the animals died days or weeks after the cessation of exposure.
4.3 SYMPTOMS AND PATHOLOGICAL EFFECTS
4.3.1 Oral Administration
Signs of intoxication in rabbits and rats dosed orally with hex in
the Treon, et al., (1955) acute toxicity studies included diarrhea,
lethargy, and retarded respiration rate. The odor of the compound could
also be detected in the feces of the animals and on their bodies ,
presumably from fecal contamination.
Among the rabbits who died, diffuse degenerative changes were seen in
the brain, heart, liver and adrenal glands. Degenerative changes were
also seen in the epithelium of the renal tubules and the lungs of these
animals were congested and edematous. The same types of degenerative
changes were also noted in the rats. In addition, some of the rats showed
acute necrotic gastritis in the stomach. Interestingly, those animals
which survived the oral tests and were sacrificed some time later
exhibited residual degenerative changes of the type described above. This
suggests that the pathological changes are semi-permanent; the severity
-------
of the lesions did diminish as the length of the post-exposure survival
interval increased, however.
Hooker Chemicals and Plastics Corporation commissioned a series of
toxicologic studies of hex. This work, performed by Industrial Bio-Test
Laboratories (IBT) consisted of 4 separate investigations: (1) a 90-day
oral toxicity study (rats); (2) a 28-day subacute dermal toxicity study
(rabbits); (3) an acute vapor inhalation study (rats); and (4) a 28-day
subacute vapor inhalation study. Since these studies focused primarily on
symptoms and toxic manifestations/effects, (rather than establishing or
documenting toxic levels) they are reported in this section.
It should be noted that we were unable to obtain the original reports
of IBT's test results, and the following summary is based on information
contained in a review document on hex prepared by Equitable Environmental
Health (1976) under contract to Hooker Chemical and Plastics Corporation,
rather than the actual IBT test results. While we believe this
information to be accurate, we do not assume responsibility for any
errors which may have been committed by EEH in interpreting the results
of the IBT results. As reported by EEH, IBT conducted a 90-day subacute
oral toxicity study, again using albino rats. Hex was mixed into the
animals food at concentrations of 0, 30, 100 or 300 ppm. No effects were
seen in any of the parameters measured: growth, food intake, mortality,
abnormal behavior, hematology, clinical blood studies, and urinalysis.
Gross pathologic examination also failed to reveal any abnormalities
which could be attributed to ingestion of hex. Similarly, organ weights
and ratios and microscopic examination of tissues and organs failed to
show treatment - related abnormalities.
Naishstein and Lisovskaya carried out another chronic toxicity
experiment on 90 white rats weighing 100-120 grams. The dosages employed
were 0.002, 0.0002, and 0.00002 mg/kg (0.04, 0.004 and 0.0004 mg/liter).
The first dose was 30 times greater than the threshold concentration with
respect to aftertaste and smell; the second dose corresponded to the
practical limit of detection by smell and the third dose was 10 percent
of the second. No deviations were observed in the behavior of the rats or
in their weights throughout the 6 month experimental period. Likewise, no
significant changes in hemoglobin, red blood cells, white blood cells or
peripheral reticulocyte counts in the experimental group as opposed to
the controls. Among animals receiving the highest dose, 0.002 mg/kg,
neutropenia and a tendency toward lymphocytosis were noted. Peripheral
blood studies of animals at the lower levels showed no abnormalities
however.
Other parameters, including tests for behavioral alterations (testing
of conditioned reflexes) were studied, however, no conclusions were drawn
from this data as it was considered by the investigators to be unreliable.
46
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4.3.2 Dermal Application
Treon, et al. , (1955) showed that dermal application of very low
dosages of hex (0.25 ml/kg) were extremely irritating and induced local
discoloration and edema. The skin became hard, encrusted and fissured
several days after application. The extent of the local damage varied
directly with the size of the dose applied. At autopsy rabbits exhibited
visceral lesions similar in appearance to those seen after oral
administration of hex. Again, diffuse degenerative changes were seen in
the brain, heart, adrenals, liver cells, and kidney tubules. Pulmonary
hyperemia and edema were also noticed. Animals killed 7-21 days post-
application of the compound showed evidence of the same type of
degenerative changes.
Monkeys dosed with various concentrations of hex in solution
exhibited discoloration of the skin which increased directly as the
concentration of hex applied increased. Swelling, oozing and encrustation
similar to that described above for rabbits was seen. Eventually healing
took place, but scarring and hair loss in the area of application
appeared to be permanent.
IBT also reported results of a 28-day subacute dermal toxicity study
using albino rabbits. (EEH, 1976). Hex solution was allowed to contact
the shaved, unoccluded skin of rabbits for an unlimited period of time
(test material not washed off). The test animals were dosed 5 days a week
for a period of 4 weeks, or 20 applications in all. The concentration of
hex in Group I was 0.1 per cent (weight/volume); in Group II, the
concentration was 0.5 per cent. None of the animals died and no
pharmacotoxic symptoms were noted, however, both hex solutions were
extremely irritating to the skin and slight losses in body weight
occurred in some of the rabbits receiving the higher concentration (Group
II). No adverse effects were noted in hematological studies, clinical
blood chemistry studies, and urinalysis. No significant gross or
microscopic pathology was noted, except of course, the local skin,
lesions. Gross skin changes were characterized by fibrosis, escharosis
(scarring) and slight-to-severe desquamation. Microscopic examination
revealed acanthosis and hyperkeratosis involving the epidermis. This
effect was seen in a few of the animals in Group I and most of the
animals in Group II. Such findings were attributable to the irritant
action of the test compound.
4.3.3 Inhalation Tests
Animals who survived the vapor exposure sessions lost weight and many
of these animals still had not regained their initial weights as long as
6 to 8 weeks after inhalation of hex.
Animals (rats, rabbits, guinea pigs and mice) exposed to vapors of
hex showed signs of extreme irritation of the eyes and mucous membranes
(Treon, et al. , 1955). At very high concentrations (46.5 ppm) animals
responded by rubbing their noses with their forefeet, closing their eyes
-------
and retracting their heads. This was accompanied by sneezing, tearing and
irregular breathing. In less than 30-60 minutes the animals were gasping
for breath.
Lower concentrations of hex vapor (12.4 and 13-8 ppm) produced
similar irritation of the mucous membranes although somewhat milder in
degree. The same symptoms were even seen at the very low dosages 1.0 and
1.6 ppm, however, the symptoms developed over a period of hours, rather
than minutes. Exposure to very low concentrations (0.33 ppm and 0.15 ppm)
resulted in some irritation of the eyelids and increased respiratory
rate, however, in the case of the latter dosage (0.15 pm) irritation was
only in the mice, which developed mild respiratory changes (Treon, et
al., 1955).
At autopsy, degenerative changes similar to those described from oral
and dermal administration were seen in all species of animals tested.
Prolonged intermittent exposure to as little as 0.15 ppm hex vapor
concentrations induced slight degenerative changes in the livers and
kidneys in all species of animals employed.
Industrial Bio-Test Labortories (IBT) also conducted two vapor
toxicity studies: an acute test and a 28-day subacute test (EEH, 1976).
In the acute vapor toxicity tests, Charles River rats were exposed for 4
hours to varying concentrations of hex in air. An acute LC 0 of 3.67 ppm
(0.041 mg/1 of air) was reported. Complete necropsies were performed at
death for those who died shortly after exposure and at the termination of
the study for those who survived. Acute pneumonia was observed in the
rats which died following exposure to the test material. Treated rats
that survived to the end of the study showed emaciation and chronic
proliferative inflammatory changes in the lungs.
The second IBT test consisted of a 28-day subacute vapor inhalation
study using albino rats. Two groups of 10 rats each were exposed to hex
vapor for 6 hours per day, 5 days per week for 4 weeks. A third group of
ten rats served as untreated controls. One group (Group I) was exposed to
vapor containing 0.529 ppm hex (0.006 mg/1) and the other experimental
group. (Group II) was exposed to 1.23 ppm (0.014 mg/1). Hematologic and
clinical chemistry studies and urinalysis were performed on days 0 and
28. On the 28th day the animals were sacrificed. EEH made no mention of
mortality in either exposure group, so presumably all animals survived
until the time of sacrifice. Neither hematologic nor clinical studies or
urinalysis revealed any abnormalities directly attributed to the test
material vapor. Statistical analysis did reveal increases in absolute
liver weight and liver: brain weight ratios among the rats exposed to
1.23 ppm hex vapor. Gross pathological examinations failed to reveal
abnormalities attributable to the test compound; microscopic examination
of tissue revealed hepatocytomegaly and necrotizing hepatitis. These
effects were thought to be attributable to hex exposure.
An early study by Ingle (1953) showed that previous observations of
vapor toxicity of chlordane could be explained on the basis of unreacted
-------
ingredients in earlier chlordane formulations. Chief among these
unreacted materials was hex, which caused samples of chlordane to give
off irritating volatiles. This problem had been eliminated by 1953 by the
more complete removal of unreacted ingredients in the pesticide. Ingle
(1953) tested the hypothesis that hex may have been the actual agent
responsible for high rates of mortality among mice in previous chlordane
tests by replicating an earlier study using relatively pure chlordane and
chlordane mixed with hex in varying (2.5-10.0 percent) proportions. The
actual concentration of hex in the exposure chamber was not reported.
Albino mice exposed to air passing through a saturation train containing
the test solutions exhibited the same toxic symptoms and high mortality
with the exception of the "control" group exposed only to chlordane.
Onset and severity of symptoms were directly proportional to the volume
of added hex.
Gross pathologic findings in the organs of the mice included
hemorrhagic areas in the lungs and lesions of unspecified type in the
liver. Microscopic findings included congestion of capillaries and edema
of the lungs, coagulative necrosis, hyalinization , bile duct
proliferation and cytoplasmic oxyphilia. Kidney damage included protein
leakage, degeneration of the tubular epithelium and capillary engorgement
in the glomerular tufts. Extent of tissue injury was proportional to the
volume of added hex. Thus, Ingle concluded that previously reported vapor
toxicity to mice should not have been attributed to chlordane, but rather
to an unreacted intermediate, namely hex.
4.i» COMPARATIVE TOXICITY
Treon, et al., (1955) found hex to be more toxic than either phosgene
or carbon tetrachloride. Based on acute vapor toxicity to rabbits, hex
was found to be considerably more toxic, based on comparable atmospheric
concentration. Whereas less than 1/2 of a group of rabbits died following
exposure to 75-80 ppm phosgene for 30 minutes, exposure of rabbits to 7.2
ppm hex produced death in 1/3 of the test animals. Exposure to the same
concentration for 60 minutes was uniformly lethal.
On the basis of actual toxicity, hex in the concentration of 0.15 ppm
is roughly comparable to carbon tetrachloride at 100 ppm.
4.5 METABOLISM
Only two studies which address the metabolism of hex could be located
(Mehendale, 1977; Kommineni, 1978). The latter study focuses upon
absorption and elimination of hex while the Mehendale study is more
concerned with the disposition of hex within the body and modes of
elimination.
The Kommineni study consisted of two parts. The first consisted of a
study of rats exposed to various doses of hex by gavage while the second
portion examined guniea pigs exposed to varying doses of hex via dermal
application. Inferences regarding patterns of absorption, metabolism, and
-------
excretion are based on gross pathology findings and histopathologic
findings at necropsy.
In the first series, a total of 10 female rats were exposed to 0, 50,
100, 150, 200, and 300 mg/kg of hex by gavage. All animals were
sacrificed 24 hours post-treatment. The rats were necropised and lungs,
liver, spleen, kidneys, adrenals, heart, stomach, and intestines were
saved for histopathology evaluation.
Gross pathology of the rats exposed to 200 and 300 mg/kg revealed
brown discoloration around the nostrils and anus of the rats. The urinary
bladders of two of the four rats contained brown fluid. Subserosal
emphysema of the nonglandular stomach was evident in one animal. On
histopathologic examination, the lungs showed atelectasis with moderate
thickening of the alveolar walls. The alveolar walls contained moderate
numbers of macrophages and neutrophils. Some bronchi contained denuded
epithelium. No edema was present in the lungs. Rats receiving lower
dosages showed similar, but milder, changes. The stomachs of rats
receiving dosages of 200 or 300 mg/kg showed coagulative necrosis of the
gastric squamous epithelium. The submucosa of the nonglandular part of
the stomach (submucosa, submuscularis, muscularis) showed moderate edema.
Epithelium of the glandular part of the stomach showed no
treatment-related changes. Animals receiving lower doses showed similar
changes in the stomach. Ulcers of the nonglandular portion of the stomach
were seen in several of the animals. At all dosages, the other organs
were unremarkable.
The author commented that these morphological changes indicate that
hex is absorbed through the squamous epithelium of the nonglandular part
of the stomach and that the major route of elimination of hex is through
the lungs.
In the second part of the study, four male guinea pigs were painted
on the skin (site unspecified) with hex at dosages of 0, 300, 600, and
1200 mg/kg and sacrificed 24 hours after the exposure. All animals were
necropsied and the lungs, liver, pancreas, kidneys, adrenals, urinary
baldder, heart, skin, stomach, and intestines were saved for
histopathologic evaluation.
On gross pathology, subcutaneous edema was seen extending from the
inguinal area to the sternum. At the lowest dosage, the lungs were highly
expanded and showed rib impressions on the parietal surface. Similar but
more severe changes were seen in the animal receiving 600 mg/kg. The
animal painted with 1200 mg/kg expired prior to sacrifice; the trachea
was filled with frothy fluid. Histopathologic examination of the lungs
revealed atelectasis with thickened alveolar walls containing moderate
numbers of macrophages and neutrophils. Intense congestion of all
pulmonary blood vessels and occasional alveolar edema was seen in the
animal receiving the 1200 mg/kg dose. In the skin, moderate to marked
edema disrupted the collagen bundles. Focal pockets of neutrophils were
seen in the edematous dermis. Edema extended throughout the thickness of
50
-------
the adipose tissue layer. One animal showed partial thrombosis of medium
size veing situated deep in the dermis. The skin appendages were normal.
Kommineni concluded, "Hex is absorbed through the skin and probably
is eliminated through the lungs. Unlike the rat stomach, the squamous
epithelium of the guinea pig skin and its adnexa did not show necrotic
changes. This is probably due to two factors, surface area and transit
time".
In the Mehendale (1977) study, radiolabeled hex was administered by
oral intubation to four male Sprague-Dawley rats in order to examine
absorption, metabolism, and ..excretion of the compound following a single
oral dose. After dosing with C-hex (5 u moles, 1 uCi per animal), the
rats were maintained in metabolism cages for 7 days, during which daily
urine and fecal samples were collected. After 7 days, the animals were
sacrificed and the major organs were removed and radioassayed.
14
Urine and powdered fecal samples were radioassayed for total C. An
average of approximately 33 percent of the total dose was excreted in the
urine after 7 days. About 87 percent of that (approximately 28.7 percent
of total dose) was eliminated during the first 24 hours after the
administration of the compound. Fecal excretion accounted for 10 percent
of the total dose; nearly 60 percent of the 7-day fecal excretion
occurred during the first day.
Beyond -the third day after treatment, only trace amounts of the
hex-derived C were eliminated in the feces. Tissues retained only trace
amounts of hex after 7 days. For example, the kidney retained only about
0.5 percent of the total dose and the liver less than 0.5 percent. Other
organs and tissues--fat, lung, muscle, blood, etc.—contained even less
of the radiolabel. Such findings suggest that at least half of the
administered hex was eliminated by routes other than urine and feces. The
author felt that the respiratory tract is probably the major route of
excretion.
The nature of the radioactivity excreted in the urine was examined
searching for possible metabolites, it was found that about 70 percent of
the radioactivity in the urine was extractable using a hexane:isopropanol
(9:1) mixture. The organic solvent was concentrated, applied to
thin-layer chromatography (TLC) plates, and developed in three solvent
systems. The radioactive spots were visualized by auto-radiography on
medical x-ray film. The results suggested the presence of at least four
metabolites; however, at the time of this writing they had not been
identified and characterized.
14
Disposition and biliary excretion o£ C-hex was studied by injection
of approximately 1 uCi (5 u mole) of C-hex into the femoral vein of
anesthetized rats. Timed samples of blood and bile were collected for 1
hour from the femoral artery and common bile duct which had been
cannulated prior to dosing. Approximately 9 percent of the administered
dose was excreted in the bile in 1 hour. Because this quantity is
51
-------
equivalent to that excreted in the feces over 7 days, enterohepatic
circulation of this compound is probable. The nature of the compound
present in the bile is not yet known.
At the end of the above experiments, the animals were sacrificed and
the liver and kidneys were removed. Tissue homogenates from the organs
were radioassayed and the distribution of the radioactivity among the
various subcellular fractions was examined by assaying the various
centifugation fractions. Kidney cytosol accounted for 93 percent of the
radioactivity in the total kidney homogenate. This behavior is consistent
with rapid urinary excretion. Similary, 68 percent of the radioactivity
in the liver homogenate was associated with the liver cytosol fraction,
once again consistent with rapid excretion.
Pre-exposure of some of the rats to hex (50 mg/kg/day) for 3 days
prior to the experiment did not affect blood decay curves and biliary
excretion; however, an ^increased concentration in the kidneys after a
single challenge with C-hex was observed.
Whitacre (1978) reported that Vesicol has. contracted an independent
metabolism study in rats and mice using C-hex. The metabolism of hex
was determined both after single acute dosing and repeated administration
over a period of about 30 days. The results of these studies have not yet
been officially reported although verbal appraisal of some results has
been provided to Velsicol.
It appears that results of this study do not agree closely with the
Mehendale study. The recent study shows hex to be eliminated from mammals
(mice and rats) mainly by the fecal route and with no more than about 15
percent being eliminated in urine. Further, these studies do not indicate
any significant amounts of pulmonary elimination of hex or its
metabolites. Whitacre (1978) believes that the poor recoveries in feces
in the Mehendale study may be the result of volatility of hex or its
metabolites before removal for analysis. Losses during sample preparation
undoubtedly further complicate the analysis of fecal matter.
4.6 Teratogenicity
International Research and Development Corporation (1978) has
recently completed a pilot teratology study using pregnant Charles River
(CD) rats. Negative findings with respect to teratogenic effects were
reported for oral hex dosages up to 100 mg/kg/day.
The test protocol employed in the pilot teratology study involved
administration of various dosages of hex to 30 female Charles River (CD)
rats approximately 12 weeks of age. Females were mated with male rats of
the same strain. After mating, the females were assigned to six groups,
one control and five treatment groups of five rats each. Hex was
dissolved in corn oil and administered by gavage from day 6 through day
15 of gestation. Dosage levels of 3, 10, 30, 100, and 300 mg/kg/day were
-------
administered to the test groups and the control group was given the
vehicle (corn oil) on a comparable regimen of 10 ml/kg/day.
During gestation, the females were observed for clinical signs of
toxicity, mortality, and body weight gains. They were then sacrificed on
gestation day 20 and the uterine contents examined for viable and
nonviable fetuses, early and late resorptions, and total implantations.
There were no differences in the four treatment groups given 100
mg/kg/day or less when compared to the control group in terms of number
of viable or nonviable fetuses, resorptions, implantations, or corpora
lutea. Rats receiving doses of 3 or 10 mg/kg/day showed no
treatment-related changes in appearance or behavior. Rats receiving 30
mg/kg/day or higher showed staining of the anogenital area and reduced
body weight gains. The females in the 100 mg/kg/day group had body weight
losses during the first 3 days of treatment and reduced weight gains for
the remainder of the study. Survival was 100 percent for all rats given
100 mg/kg/day or less. All rats in the 300 mg/kg/day group were dead by
gestation day 10.
Various reproductive parameters examined in the pilot teratology
study are shown in Table 4.2
4.7 Mutagenicity
Hex has been tested for mutagenicity and reported non-mutagenic in
both short-term in vitro mutagenic assays (National Cancer Institute,
1977; Industrial Bio-Test Laboratories, 1977; Litton Bionetics, 1978a)
and in a mouse dominant lethal study (Litton Bionetics, 1978b).
The National Cancer Institute (1977) reported that preliminary
results indicated that hex was non-mutagenic in Escherichia coli K12
(mutation site not specified) in the presence of a mammalian metabolic
activation system containing mouse liver microsomes.
Negative results were also reported by Industrial Bio-Test
Laboratories (1977) using a test protocol almost identical to the Ames
Mutagenic Assay (Ames, et al. 1975). The tests used four or five strains
of Salmonella typhimurium with and without metabolic activation. Hex was
dissolved in acetone and added to the microbial assay plates in dosages
from 10-5000 ug/10 ul. Concentrations greater than 10 ug/10 ul produced a
bacteriocidal effect in three of the strains tested; a posible lethal
effect occurred at 2500 ug/10 ul or greater in the fourth strain. A
repressive effect was noted in three of the strains at concentrations
below 10 ug/10 ul. Volatilate (volatile vapors) of hex were also tested
on one strain using the vapor from hex concentrations of up to 2500 ug/10
ul and exposure times of up to 2 hours. Results from two successive
assays in the absence of rat liver enzymes (hex concentrations 10, 25,
50, 75, and 100 ug/10 ul) were negative in all four tester strains. Two
assays using the same dosages in the presence of rat obtained for the hex
effusate as well. The investigators expressed concern over the repressive
effective of hex on the test bacteria, stating "It appears that hex is
53
-------
TABLE 4.2. PILOT TERATOLOGY STUDY IN RATS: CAESAREAN
SECTION DATA FOR INDIVIDUAL FEMALES.3
Dosage Level
Dam Number
Control:
73662
77334
77336
77428
77428
Total
Mean
3 Big/kg/day.
73642
77342
77343
77426
77428
Total
Mean
10 mg/kg/day:
77304
77309
77346
77427
77436
Total
Mean
30 mg/kg/dav:
77310
77313
77350
77438
77450
Total
Mean
100 mg/kg/day:
73673
77302
77314
77415
7743S
Total
Mean
300 mg/kg/day:
73758
77324
77333
77417
77445
Viable Nonviable
Fetuses Fetuses
13
14
12
11
15
65
13.0
16
17
16
12
15
76
15.2
17
13
11
12
15
68
13.6
14
13
6
11
12
56
11.2
16
14
15
11
12
68
13.6
Died,
Died,
Died,
Died,
Died,
Source: International
0
0
0
0
0
0
0.0
0
0
0
0
0
0
0.0
0
0
0
0
0
0
0.0
0
0
0
0
0
0
0.0
0
0
0
0
0
0
0.0
gestation day
gestation day
gestation day
gestation day
gestation day
Research and
Late
Resorptions
0
0
0
0
0
0
0.0
0
0
0
0
0
0
0.0
0
0
0
0
0
0
0.0
0
0
0
0
0
0
0.0
0
0
0
0
0
0
0.0
9 - gravid
10 - gravid
10 - gravid
10 - gravid
10 - gravid
Development Cc
Plant
Early Implantation Implan- Corpora
Resorptions Loss tations Lutea
1
1
1
1
0
4
0.8
0
0
0
1
0
1
0.2
0
0
3
0
0
3
0.6
0
1
0
0
0
1
0.2
0
0
2
0
0
2
0.4
>rp. (1978)
1
1
1
1
0
4
0.8
0
0
0
1
0
1
0.2
0
0
3
0
0
3
0.6
0
1
0
0
0
1
0.2
0
0
2
0
0
2
0.4
14
15
13
12
15
69
13.8
16
17
16
13
15
77
15.4
17
13
14
12
15
71
14.2
14
14
6
11
12
57
11.4
16
14
17
11
12
70
14.0
14
15
13
22
16
80
16,0
16
17
16
18
15
82
16,4
18
13
14
13
15
73
14,6
14
16
7
14
14
65
13.0
16
14
17
11
12
70
14.0
-------
probably non-mutagenic and that some toxic effect prevailed with respect
to the tester strains required for this assay. Analysis of variance and
multiple comparison of the data confirms this "observation".
Litton Bionetics (19?8a) conducted a mouse lymphoma cell assay in
order to evaluate the capability of hex in inducing specific locus
forward mutation. The indicator cells used in the assay were Fischer
mouse lymphoma cells derived from cell line L5178Y. These cells are
heterozygous for a specific autosomal mutation at the TK locus and are
bromodeoxyuridine (BUdR) sensitive. Scoring for mutation is based on
selecting cells which have undergone forward mutation from a TK+/-to a
TK-/- genotype by cloning them in soft agar with BUdR. Cells were
maintained in Fischer's medium for leukemic mouse cells with 10 percent
horse serum and sodium pyruvate. The dosages used in the test were
predetermined by exposing the cells to a wide range of hex concentrations
and measuring the reduction of growth potential following a 4-hour
exposure at each dose. The maximum dose selected was that which produced
a 50 percent reduction in growth. The actual hex dosages employed were:
0.00040, 0.00048, 0.00056, 0.00064, and 0.00125 ul/ml in the activated
series (mouse liver microsomes were added to the growth medium). A
nonactivated series using somewhat lower dosages was included also.
Both negative and positive controls were used; the negative control
for both series was the solvent dimethylsulfoxide (DMSO), whereas
ethylmethanesulfonate (EMS) and dimethylnitrosamine (DMN) were used as
positive controls in the nonactivated and activated systems,
respectively. Hex was added to the cells in the growth medium for 4
hours. The cells were then washed and allowed to express in the growth
medium for 3 days. After the expression period, results were evaluated by
counting the TK-/-mutants after cloning the cells in a selection medium
(soft agar with BUdR).
Hex dissolved in DMSO was evaluated over the concentration range of
0.0000025 ul/ml to 0.00125 ul/ml. Considerable toxicity occurred at
concentrations greater than this and the extent varied according to the
presence of the mouse liver activation system as shown in Table 4.3. No
cells treated with hex (at the concentrations shown) survived in the
non-activated system.
Hexachlorocyclopentadiene did not induce forward mutation in L5178Y
cells. The data presented in Table 4.3 show the concentrations of the
test compound employed, the number of mutant clones obtained, surviving
populations after the expression period, and calculated mutation
frequencies. No dose-related trends in either absolute number of mutants
or mutant frequencies were observed, and at no level did any of the test
parameters increase significantly over the spontaneous level.
Consequently, hex was considered to be nonmutagenic under the conditions
of this assay.
55
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TABLE 4.3. SUMMARY OF MOUSE LYMPHOMA (L5178Y) RESULTS.
a,b
vn
TEST0
NONACTIVATION
Solvent Control
Negative Control
EMS 0.5 yl/ml
ACTIVATION
Solvent Control
Negative Control
DMNS 0.5 yl/ml
Test Compound
0.00002 yl/ml
0.00004 pi/ml
0.00008 ul/ml
0.00016 ul/tnl
0.00032 yl/ml
S-9
Source Tissue
—
—
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
Mouse
—
—
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Liver
Daily Counts
(Cells/ml x 10ES)
1
16.8
13.2
9.0
15.2
14.2
7.2
16.8
13.0
12.4
13.6
18.2
2
10.2
12.0
9.2
9.6
13.0
7.6
9.0
12.4
9.8
13.8
9.0
3
13.8
15.0
11.8
13.2
10.6
8.2
10.6
9.6
16.2
7.4
10.0
Relative
Suspension Total
Growth (% Mutant
of Control) Clones
100.0
100.5
41.3
100.0
101.6
23.3
83.2
80.3
102.2
72.1
85.0
48.0
48.0
597.0
55.0
39.0
322.0
99.0
50.0
55.0
45.0
38.0
Total
Viable
Clones 1
257.0
234.0
89.0
281.0
293.0
55.0
288.0
269.0
194.0
359.0
309.0
Relative
Cloning Percent Mutant
Efficiency Relative Frequency
[ % of control) Growth X 10E-6
100.0
91.1
34.6
100.0
104.3
19.6
102.5
95.7
69.0
127.8
110.0
100.0
91.5
14.3
100.0
105.9
4.6
85.3
76.9
70.6
92.1
93.5
18.7
20.5
670.8
19.6
13.3
585.5
34.4
18.6
28.4
12.5
12.3
Source: Litton Bionetics, Inc. (1978a).
Hexachlorocyclopentadiene dissolved in dimethyl sulfoxide.
A
Concentrations are given in microliters (ul) per milliliter.
Relative suspension growth x relative cloning efficiency/100.
Mutant clones/viable clones x 10E~ .
Ethylmethanesulfonate.
^imethylnitrosamine.
-------
The mutagenic properties of hex were also evaluated in a dominant
lethal study of mice (Litton Bionetics, 19?8b). The dominant lethal assay
provides a means of determining whether a compound is capable of inducing
damage in the germ cells of treated male mice. Dominant lethality is
manifested in various forms of fetal wastage, both pre- and
post-implantation. Positive dominant lethal assays indicate that a
compound is able to reach the developing germ cells. Chromosome
aberrations including breaks, rearrangements, and deletions as well as
ploidy changes and nondisjunction are believed to produce positive
results on this test. Since substances capable of producing gross
chromosomal lesions are probably capable of producing more subtle
balanced lesions or specific locus mutations, the test also provides
suggestive evidence of nonlethal mutations transmissible to future
generations as well.
Litton Bionetics reported negative results, that is, there was no
evidence of significant dominant lethal activity by hex in mice. The test
protocol called for the assignment of ten random bred male mice to one of
five groups. Three test groups received hex dosages of 1.0, 0.3, and 0.1
mg/kg, respectively. These dosages were determined by deriving an LD5
level (1.0 mg/kg) and taking one-third and one-tenth of that dose. A
fourth group received only the solvent and the fifth group served as a
positive control. Hex was mixed in the feed of the three experimental
groups and the solvent control group for five consecutive days. The
positive control group received a known mutagen, triethylenemelamine
(TEM) in a single intraperitoneal injection. Two days following
treatment, each male was caged withh two unexposed virgin females. At the
end of seven days, these females were removed and replaced by two
unexposed virgin females. This mating cycle was continued for seven
weeks. Each pair of female mice was killed two weeks after mating and
necropsied. Their uterine contents were examined for dead and living
fetuses, resorption sites, and total implantations. All test parameters
(fertility index, average implantations per pregnancy, average
resorptions (dead implants) per pregnancy, proportion of females with one
or more dead implantations, proportion of females with two or more dead
implantations, and the ratio of dead implantations to total implantations
were within normal limits based on historical concurrent control levels
for this test. Thus, there was no evidence of dominant lethal activity in
any of the hex treated groups. The positive control group, however, did
show the expected dominant lethal activity.
4.8 CARCINOGENICITY
Various types of evidence may be used in evaluating the possible
carcinogenic activity of a substance. In order of preference, these
include: (1) human data; (2) animal data; (3) short-term (in vitro)
tests; (4) metabolic pattern; and (5) structure-activity relationships.
This section summarizes what is known about each of the above.
No epidemiologic studies or case reports examining the relationships
between exposure to hex and cancer incidences could be found in the
57
-------
literature. Hooker Chemicals and Plastics Corporation reports that an
in-house study of the mortality patterns of hex-exposed workers is now
underway; however, the study is far from being completed (Zavon, 1978,
personal communication). Other in-house studies of workers employed in
the manufacture of pesticides (including hex) are reportedly being
conducted by Velsicol Chemical Corporation. We were unable to obtain any
further information on the current status or findings of these studies.
The National Cancer Institute concluded that toxicologic studies of
hex in animals have not been adequate for evaluation for carcinogenicity
(National Cancer Inst., 1977a). Chronic toxicity studies reported were
based on too few animals in some cases and/or the duration of the
experiments was too short for appropriate evaluation of chronic effects,
including carcinogencity (World Health Organization, 1976; National
Cancer Institute, 1969).
Only one short term in vitro test of hex for carcinogenic activity
could be identified.
Litton Bionetics (1977) reported the results of a test to determine
whether hex could induce malignant transformation in BALB/3T3 cells in
vitro. The cells and methodology of the test were those of Kakunaga
(1973)- The basic rationale of the test and its validity as an indicator
of carcinogenic activity was described by the investigators as follows:
The endpoint of carcinogenic activity is determined by the presence
of fibroblastic-like colonies which are altered morphologically in
comparison to the cells observed in normal cultures. These
(transformed) cells grow in criss-cross, randomly oriented fashion
with overlapping at the periphery of the colony. The colony exhibits
dense piling up of cells. On staining, the foci are deeply stained
and the cells are basophilic in character and variable in size. These
changes are not observed in normal cultures, which stain uniformly.
Cell cultures with very little or no spontaneous transformation are
maintained for use in these tests. The data generated at each dose level
of the test material are analyzed using the t statistic. A significant
set of data for any dose level may be sufficient to indicate a positive
response. Because this assay is still nonroutine, and definitive criteria
for evaluation have yet to be developed, scientific judgement and expert
consultation are needed for appropriate interpretation of results.
The BALB/3T3 cells used in the test were grown in Eagle's minimal
essential medium (EMEM) supplemented by 10 percent fetal calf serum.
Cultures were passaged weekly in 60 mm culture dishes. Approsimately
10,000 cells were seeded into 50 ml sterile tissue cuture flasks and
incubated in EMEM to permit attachment. After the cells were attached,
the control and test compounds were added to the plates. Dosages of
0.00001, 0.00002, 0.000039, 0.000078, and 0.000156 ul/ml of hex were
employed. The maximal dosage, 0.000156 ul/ml, was determined by selecting
from preliminary cytotoxicity tests the maximum dosage which permitted
-------
survival of at least 80 percent of the cells. 3-methylcholanthrene at 5
ug/ml was used as a positive control and the test compound solvent was
used as a negative control. Ten replicates per dose level were prepared
and chemical exposure was maintained for 48 hours. Plates were then
washed free of the compound and replenished with fresh growth medium. The
plates were then incubated for an additional 3-4 weeks with twice-weekly
medium changes. Cell integrity was monitored by daily observations. Cells
were separated from the medium, washed with saline, and stained. They
were examined for stained foci; all potential foci were examined
microscopically. Results were presented as the number of foci per set of
replicate plates at each dosage level.
The test material was quite toxic to cells as indicated in the
preliminary range-finding tests. No significant carcinogenic activity for
hex was reported under the conditions of this test. A low level of
spontaneous transformation was observed on all of the plates. Only the
3-methylcholanthrene treated plates showed a significantly higher number
of transformed foci than the negative control.
It should be noted that in this and other cell culture tests,
extremely low dosages of hex were used. Because hex is relatively toxic
to cells in culture and test protocols normally require a high survival
rate, the applicability of test results to environmental conditions is
unclear. Taken together, however, the mutagenicity and carcinogenicity
tests conducted by Litton (1977, 1978a) suggest that outright toxicity,
rather than chronic effects, is perhaps the critical effect of hex, even
at very low dosages. Extremely poor survival has also been problematic in
several subchronic tests of hex in mammalian species.
A very recent study involving chronic dietary exposure of rats to
hexachlorobutadiene also provides some insight into the relationship
between direct toxic effects and chronic effects (i.e., carcinogenesis)
in this related compound (Kociba, et al., 1977). Male and female
Sprague-Dawley rats were maintained on diets supplying 20, 2.0, 0.2, or 0
mg/kg/day of hexachlorobutadiene (C-46) for up to 2 years. Rats ingesting
0.2 mg/kg/day had no discernible ill effects that could be attributed to
this dose level. Ingestion of the intermediate dose level of 2.0
mg/kg/day caused some degree of toxicity, affecting primarily the kidney
in which increased renal tubular epithelial hyperplasia was noted.
Urinary excretion of coproporphyrin was also increased at this dose
level. Ingestion of the highest dose level of 20 mg/kg/day caused a
greater degree of toxicity. Effects included decreased body weight gain
and length of survival, increased urinary excretion of coporporphyin,
increased weights of kidneys, and renal tubular adenomas and
adenocarcinomas, some of which metastasized to the lung. In this study
irreversible toxicological effects, such as the developmment of
neoplasms, occurred only at a dose level which caused significant tissue
injury and other manifestations of toxicity. No neoplasms resulted with
dose levels which caused no injury or only mild, reversible injury.
59
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Little information is available on the metabolism of hex. Although at
least four metabolites were found in the Mehendale (1977) study, at the
time of this writing they had not been identified. Thus, the metabolic
pathway is uncertain.
As far as structure/activity relationships are concerned, the
National Cancer Institute (1977a) speculated that as a cyclopentene vinyl
halide, hex potentially may be metabolized to an electrophile. In
addition, hex is related to the pesticides dieldrin, heptachlor, and
chlordane which have been found to induce liver tumors in mice following
oral administration (National Cancer Inst., 1977b; 1977c).
Hex has recently been selected for testing in the National Cancer
Institute's test program (National Cancer Inst., 1977a). The reasons
given for its selection include: (1) its high potential for exposure (as
an industrial intermediate used in the manufacture of pesticides, flame
retardants and dyes, Pharmaceuticals, resins, and germicides); (2) its
suspect chemical structure; and (3) the relative lack of information on
the effects of chronic exposure to this compound.
60
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5.0 HUMAN TOXICITY
Very little information is available concerning the effects of hex
exposure on humans. Unfortunately, no systematic epidemiologic
investigations of the toxicity of this compound have been reported.
Nevertheless, several reasonable inferences might be made on the basis of
animal studies. From the early studies of Treon, et al., (1955) it is
apparent that hex is an extremely potent irritant and is toxic by all
these major modes of exposure; oral, dermal and inhalation. Furthermore,
animal studies indicate that the relative toxicities of hex via oral and
dermal exposures are remarkably similar. It is presently unknown whether
oral exposure (e.g. through ingestion of hex-contaiminated drinking
water) constitutes a significant source of human exposure. Oral contact
does not appear to be a likely mode of occupational exposure. However,
dermal and inhalation exposures to hex might be anticipated among workers
engaged directly in hex manufacture or in formulation of other related
pesticides where it may be present as an impurity. A recent incident in
which scores of workers at a sewage treatment plant in Louisville,
Kentucky, experienced a variety of toxic symptoms following the improper
disposal of hex manufacturing wastes has created a great demand for
information concerning the effects of hex exposure on humans. This
episode is described in greater detail later in this section.
It is essential that persons having opportunities for skin contact
with hex should be equipped with, and trained in the use of appropriate
protective clothing and respiratory protection. The product bulletin on
hex (Velsicol, 1976) states that skin contact should be avoided and
persons handling hex should be outfitted with Neoprene gloves and
protective goggles and face shields. Adequate protective clothing should
be worn at all times.
5.1 DETECTION THRESHOLDS
According to Treon, et al., (1955) a very faint odor of hex was
detectable in air by some individuals at concentrations as low as 0.15
ppm (.0017 mg/1) which was the lowest concentration employed in their
experiments. At approximately twice that concentration (0.33 ppm) a very
pronounced, pungent odor was present.
Treon, et al., observed that headaches developed among laboratory
workers following incidental exposure to hex vapor from the respiratory
chambers used for their vapor inhalation experiments. The exact
concentration of hex escaping into the laboratory from the opening of the
respiratory chamber is unknown, however the chamber was not opened until
the contaminated air had been exhausted and the chamber flushed for some
time with clean air. Thus, the ambient concentration producing headaches
61
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among the laboratory workers was well below the doasges employed in the
animal experiments. Because no mention is made of any other irritative
symptons, (e.g. lacrimation, etc.) it seems reasonable to speculate that
the concentration of hex present was somewhere in the range between 0.15
ppm - 1.0 ppm, above the detection threshold but below the level
producing acute symptoms of irritation.
Irritant effects are elicited at a vapor concentration greater than
that shown to produce chronic toxicity in animals. Thus, Treon, et al.,
concluded that the irritant effects of hex vapors are not sufficiently
pronouced to serve as a warning that a hazardous level of hex vapor is
present and/or that hazardous exposure is taking place.
According to Naishstein and Lisovskaya (1965) hex may be detected by
taste and smell at very low concentrations in water. The threshold level
for altering the organoleptic qualities of water was placed at 0.0014 -
0.0010 mg/1 by these investigators.
The toxicity of hex has some implications for standards setting.
Prolonged intermittent exposure of animals to hex vapors at
concentrations as low as 0.15 ppm induced slight degenerative changes in
several organ systems. No overt signs of toxicity were noted at the time
of exposure, however, the pattern of exposure employed in Treon, et al's
(1955) low dose, chronic toxicity studies represents a reasonably close
approximation to an occupational exposure pattern, (e.g. exposure to 0.15
ppm for 7 hrs. per day, 5 days per week). Although systematic
observations of workers exposed chronically to known concentrations of
hex are necessary to establish safe limits of human exposure, Treon, et
al., concluded that, "at the very least, it would seem unwise to expose
workers to even the least severe of the exposure conditions (0.15 ppm)
unless or until there is some basis in terms of human experience. Men
exposed to the vapor of this chlorinated hydrocarbon, even for short
periods of time, should have faultless respiratory protection."
In keeping with this, Velsicol's Product Bulletin for hex (Velsicol,
1976) states that, "vapors of hex should be avoided, and adequate
ventilation should always be provided when hex is handled in an enclosed
area. Self-contained air masks, or full face gas masks having canisters
of the 'acid gases and organic vapors' type should be available at all
times for emergency use (e.g. spills)."
The present Threshold Limit Value (TLV) for industrial exposures to
hex is set at 0.01 ppm as a time-weighted average over an 8 hour workday
(ACGIH, 1977). This value represents about 1/15 or 7 percent of the
lowest vapor concentration shown to produce chronic toxic effects in
laboratory mammals (0.15 ppm; Treon, et al., 1955). The "safety factor"
for hex is therefore somewhat less than for many other toxic vapors.
Similarly, Naishstein and Lisovskaya, (1965) have recommended that
the maximum permissible concentration in water, based on prevention of
organoleptic effects, should be placed at approximately 0.001 mg/1.
62
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5.2 LOUISVILLE CONTAMINATION INCIDENT
The first and only documented incident of acute toxicity of hex to
humans occurred at the Morris Forman Wastewater Treatment Plant (MFWTP)
in southwest Louisville, Kentucky. The problem apparently began about the
middle of March, 1977, when an unknown chemical, later identified as a
mixture of hex and octachlorocyclopentene (C-58), began entering the
Morris Forman sewage treatment facility. An exact date of initial
appearance at the plant, and hence, the initial date of worker exposure
is unknown. However, unusual odors became evident around March 17, 1977,
There was little reaction to these odors at first, probably because
unusual odors are not uncommon at sewage treatment facilities serving
large industrialized areas.
The odor gradually intensified over the next two weeks. From March
25-28 an odoriferous, sticky material entered the plant and gummed the
bar screens in the Screen and Grit Building of the plant. Several
employees tried unsuccessfully to clean the bar screens and grit chambers
with high pressure water. Subsequent steam cleaning caused a release of a
blue gas into the atmosphere, producing severe irritation of the eyes,
nose, throat, lungs and skin of several workers. Approximately 20
employees were referred to and treated by the local hospital emergency
room. None were hospitalized (Carter, 1977; Singal, 1978).
A sample of the material from the screen and Grit Building was sent
to the U.S. EPA Laboratory in Athens, Georgia, for analysis. The primary
contaminants in the samples were identified as hexachlorocyclopentadiene,
(hex) and octachlorocyclopentene (C-58). C-58 is a waste by-product in
the manufacture of hex (See Section 2.2.2) whose toxicity is presently
unknown. Upon this identification, the Morris Forman STP was evacuated
and closed on March 29, 1977 and its entire flow, amounting to
approximately 105 million gallons per, day was diverted directly into the
Ohio River until its partial reopening in June, 1977. Analysis of a
sludge sample is shown in Table 5.1.
Estimates of the extent of contamination indicate that about 60
million gallons (25,000 tons) of hex contaminated material was present at
the Morris Forman plant. Of this, approximately 6 tons of hex and C-58
were thought to be present in the contaminated waste. U.S. EPA's analyses
revealed hex concentrations up to 1000 ppm in the sewage water at the
time of the plant closure. The route of chemical contamination was traced
to one large sewer line which passed through several heavily populated
areas. Wastewater in this sewer showed hex and C-58 in concentrations
ranging up to 100 ppm. Samples from the sewer showed air concentrations
ranging up to 400 ppb (0.4 ppm) for hex and up to 30 ppb (0.030 ppm) for
C-58. Thus, it was decided to investigate the health of not only the
workers at the sewage treatment plant, but also residents of the area
surrounding the sewer line.
A cooperative investigation was intiated involving Region IV U.S. EPA
(Surveillance and Analysis Division), Center for Disease Control
63
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TABLE 5.1 ANALYSIS OF SLUDGE SAMPLE FROM LOUISVILLE,
KENTUCKY WASTEWATER TREATMENT PLANTa'b
COMPOUND0'd CONCENTRATION, WT. PERCENT
Octachlorocyclopentene 9
Hexachlorocyclopentadiene 4
Hexachlorobenzene 0.3
Pentachlorobenzene 0.2
Octachloronaphthalene 0.4 (estimated)
Heptachloronaphthalene 0.2 (estimated)
Hexachloronaphthalene (not quantitated)
Mirex 0.007 (estimated)
aSource: Singal (1978).
Sample obtained April 2, 1972 from Screen and Grit Building, Morris
Forman Wastewater Treatment Plant.
Q
Analysis was conducted by the U.S. Food and Drug Administration, Division
of Chemical Technology, Chemical Industry Practices Branch.
The sample was analyzed using gas chromatography interphased with mass
spectroscopy for positive identification of each compound.
-------
(Atlanta, Georgia), National Institute for Occupational Safety and Health
(NIOSH), Jefferson County (Kentucky) Health Department, and the Kentucky
State Health Department.
Information on both aspects of this investigation (e.g. community
residents on the one hand and exposed workers on the other) is thus far
unpublished, but preliminary drafts of reports were made available by
Dale Morse, M.D., who headed the epidemiologic studies conducted by the
Center for Disease Control (Morse, et al., 1978), and by Mitchell Singal
of the Hazard Evaluation and Technical Assistance Branch of NIOSH, who
reported on the follow-up investigations of workers during cleanup
operations at the sewage treatment facility (Singal, 1978). Findings from
these draft reports are summarized below: however, they should be
regarded as preliminary.
5.2.1 Plant Employee Health Effects Evaluation
All plant employees who worked at the sewage plant for two or more
days from March 14-29 were identified along with all employees known to
have sought medical treatment. Health effects evaluations, including
questionnaires, physical examinations and blood and urine tests were
performed on 42 individuals who agreed to participate. The questionnaire
covered demographic information, a detailed work-area history, symptoms
or history of chemical poisoning, personal habits and other sources of
chemical exposure. Routine tests were performed on blood and urine
specimens. Additional samples were sent to NIOSH laboratories for
potential toxic chemical analyses.
Of 193 plant employees who had worked during the latter part of
March, 42 persons were interviewed and provided blood and urine samples.
This includes 24 of 29 (83 percent) of those workers who had been
previously evaluated by local physicians, 17 of 164 of other plant
employees (a 10 percent random sample) as well as 1 non-employee
accidently exposed to the contaminated sludge. In addition, 104 of the
remaining employees completed a mailed questionnaire. Overall,
questionnaire data were obtained from 145 (75 percent) of 193 total
employees.
Results of the questionnaire indicated that 75 percent of the
employees detected an unusual odor at the plant sometime during March.
The odor was reported as early as March 1, 1977, noticeably increased by
March 14 and from then steadily increased until the plant was closed on
March 29. (see Figure 5.1).
A comparison between the time of odor detection and the onset of eye
irritation, the most common sympton, showed that irritation developed on
the same day in 45 percent of individuals, within 1-5 days in 28 percent
and after 5 days in 21 percent. Only 6 percent of employees reported
onset of symptoms prior to noticing an unusual odor at the plant.
-------
2:
O
cc
60 TJ
55-
50-
45-
40-
35-
30-
25
20-
15-
10-
5-
O
I 2 34 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
MARCH
*PLANT CLOSED MARCH 29
FIGURE 5.1 Employees who noticed unusual odor at plant, by day,
Louisville, Kentucky, March 1-28*, 1977
Adapted from Morse, et al. (3.978).
66
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Eye irritation, headache and throat irritation were the most common
symptoms, with 59 percent, 45 percent and 27 percent of employees
reporting these symptoms, respectively. Data for these and other symptoms
are reported in Table 5.2. Of 41 workers physically examined, 5 had signs
of eye irritation (tearing and/or redness) and 5 had signs of skin
irritation. Abnormalities were found in laboratory analyses of some of
the workers (e.g., LDH elevations in 27 percent and proteinuria in 15
percent of those examined). These results are inconclusive, possibly
indicating a transitory abnormality or a problem with the laboratory
analysis. There were no LDH or urinalysis abnormalities found on repeat
tests run three weeks later by another laboratory. Also, no abnormalities
were reported among individuals seen at the local hospital or by the
plant physician. Thus, the validity of these laboratory test results may
be questionable. Attempts to develop a technique to isolate and identify
concentrations of hex in blood and urine specimens were unsuccessful.
Employees worked primarly in one of the eight work areas shown in
Table 5.3- Symptoms occurred in workers of all job categories and in all
work areas. Only small differences in case rates appeared by work area
although the highest attack rates occurred in workers in the primary
treatment area where the level of hex was presumably highest.
Detailed work area histories on 124 individuals during the highest
exposure period (Table 5.4) showed that "cases" occurred in all areas of
the plant. A case was defined as an individual who reported 2 or more
major symptoms (eye irritation and headaches) or 1 major symptom and 2
minor ones (sore throat, cough, chest pain, difficulty breathing, skin
irritation). Attack rates were significantly higher for individuals who
had been exposed to the screen and grit chamber (p< .0001) and to the
primary settling area (p<.02) than for workers not exposed to these
areas.
This investigation demonstrated that 64 of 145 (44 percent) of
current employees questioned at the waste-water treatment plant had
experienced headache and mucous membrane, skin and respiratory tract
irritation after exposure to airborne hex. Highest attack rates occurred
among workers in the primary treatment area where exposure was highest
and ventilation poorest. In most cases symptoms were transient, but in
some workers, they persisted for several days. This episode clearly
demonstrates the volatility of hex and its potential for having a toxic
effect on humans. Unfortunately, the long-term effects of transient
exposures such as this incident are presently unknown.
• Velsicol has recently developed a technique for the analysis of hex in
human urine with a reported detectability limit of 0.3 ppb (Whitacre,
1978).
67
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TABLE 5. 2 SYMPTOMS OF 145 PLANT EMPLOYEES
LOUISVILLE, KENTUCKY, MARCH, 197f
Symptom
With Symptom
Percent
With Symptom
Eye irritation
Headache
Throat irritation
Nausea
Skin irritation
Cough
Chest pain
Difficult breathing
Nervousness
Abdominal cramps
Decreased appetite
Decreased memory
Increased saliva
86
65
39
31
29
28
28
23
21
17
13
6
6
59
45
27
21
20
19
19
16
14
12
9
4
4
Source: Morse, et al.(1978)
TABLE 5.3. ATTACK RATES IN EMPLOYEES, BY MAIN WORK AREA3
LOUISVILLE, KENTUCKY, MARCH, 1977
Number of
Main Work Area Employees
Primary treatment
Throughout plant
Vacuum filtration
Secondary aeration
chamber
Administration and
laboratory
Final effluent
pump station
Low pressure
oxidation
Incineration
Totals
19
71
19
14
30
10
13
l]_
193
Number
Reporting
Symptoms
17
54
15
12
22
5
10
10
145
Percentage of
Employees Re-
porting Symptoms
89
76
79
86
73
50
77
5JL
75
Percentage of Cases
of Those Reporting
Symptoms
59
48
47
42
41
40
30
210
44
Source: Morse, et al(1978).
68
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TABLE 5.4. CASE ATTACK RATES IN 124 EMPLOYEES EXPOSED TO PLANT WORK
AREAS LOUISVILLE, KENTUCKY, MARCH 25-28, 1977a
CTN
MD
Number Persons
Work Area Exposed to Area
Screen and grit
Primary settling
tanks
Vacuum filtration
Secondary treatment
Sludge
Incineration
Low pressure oxidation
Administration building
Oxygen generation
38
41
36
39
32
33
37
12
18
Attack Rate
Number Cases r for Exposed,
Among Exposed Percent
29
26
12
22
17
14
16
6
8
76
63
33
56
53
42
43
50
44
Attack Rate
for Nonexposed, c
Percent X2
35
40
53
44
46
49
49
47
48
16.52
5.25
3.36
1.30
.27
.24
.19
.02
.00
d
P
io~4
.02
NS
NS
NS
NS
NS
NS
NS
Source: Morse, et al (1978).
See text for definition of a "case".
"Results of Chi-square test of significance of association between number of "cases" and the specific
work area indicated.
Significance level of the Chi-square test for the work area indicated. NS means "not significant".
-------
5.2.2 Community Survey—
CDC workers administered a questionnaire to a systematically selected
sample of residents in a 48 block area surrounding the contaminated sewer
line. One home per block was surveyed by administering a questionnaire to
the head of each household. In all, 212 occupants of the 48-block area
were surveyed. Questions were asked concerning basic demographic data,
history of unusual odors and any symptoms noted by household members
within the prior two weeks.
Results of the community survey were essentially negative. Eight of
the 212 persons (3-8 percent) reported noticing an uunusual odor at some
time during the preceding two weeks. While some of the respondents
reported symptoms compatible with hex exposure (headache 4.7 percent,
burning or watering eyes 4.7 percent) No symptom occurred at greater than
background rates. Symptoms not associated with hex were reported just as
frequently as those possibly related to exposure. Furthermore, there was
no association between symptom rates and distance from the sewer line.
Subsequent air sampling failed to show a significant ambient
concentration of hex in the sewer line area.
5.3 CARCINOGENICITY
Hex has recently been selected for testing in the National Cancer
Institutes (NCI) test program (NCI, 1977a). The reasons given for its
selection include: (1) its high potential for exposure (as an industrial
intermediate); (2) its suspect chemical structure and; (3) the relative
lack of information on the effects of chronic exposure to this compound.
TO
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
REPORT NO.
EPA-600/1-78-047
2.
3. RECIPIENT'S ACCESSION NO.
TITLE AND SUBTITLE
Reviews of the Environmental Effects of Pollutants:
XII. Hexachlorocyclopentadiene
5. REPORT DATE
]December 1978 issuing date
6. PERFORMING ORGANIZATION CODE
. AUTHOR(S)
Mary Anne Bell, Robert A. Ewing, and Gar son A, Lutz
8. PERFORMING ORGANIZATION REPORT NO,
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Battelle-Colurabus Laboratories
505 King Avenue
Columbus, Ohio 43201
10. PROGRAM ELEMENT NO.
1HA616
11. CONTRACT/GRANT NO.
G3-03-2G08
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory, Cin-OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA/600/10
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This document is a review of the scientific literature on the biological and
environmental effects of hexachlorocyclopentadiene. Inlcuded in the review are a
general summary and a comprehensive discussion of the following topics as related
to hexachlorocyclopentadiene and specific Hexachlorocyclopentadiene compounds:
physical and chemical properties; occurrence; synthesis and use; analytical
methodology; biological aspects in microorganisms, plants, wild and domestic
animals, and humans; distribution mobility, and persistence in the environment;
and an assessment of present and potential health and environmental hazards.
More than 100 references are cited. Ihe document also contains an evaluation of
potential hazard resulting from hexachlorocyclopentadiene contamination in the
enviornment and suggests current research needs.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
COS AT I Field/Group
*Pollutants
Tbxicology, Health Effects
Hexachlorocyclopentadiene
57H
STY
8. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
91
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE
81
U S GOVERNMENT PRINTING OFFICE 1979-640-079/233
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