EPA-600/3-77-066
June 1977
Ecological Research Series
CHEMICAL/BIOLOGICAL IMPLICATIONS OF
USING CHLORINE AND OZONE FOR
DISINFECTION
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
-------
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
a Scientific and Technical Assessment Reports (STAR)
7. fnteragency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences, investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 23161.
-------
EPA-600/3-77-066
June 1977
CHEMICAL/BIOLOGICAL IMPLICATIONS OF USING
CHLORINE AND OZONE FOR DISINFECTION
by
Robert M. Carlson
Ronald Caple
University of Minnesota
Duluth, Minnesota 55812
Grant Number R-800675
Project Officer
Gary E. Glass
Environmental Research Laboratory-Duluth
Duluth, Minnesota 55804
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
DULUTH, MINNESOTA 55804
-------
DISCLAIMER
This report has been reviewed by the Environmental Research Laboratory-
Duluth, 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 recommenda-
tion for use.
-------
FOREWORD
Our nation's freshwaters are vital for all animals and plants, yet our
diverse uses of water—for recreation, food, energy, transportation, and
industry—physically and chemically alter lakes, rivers, and streams. Such
alterations threaten terrestrial organisms, as well as those living in water.
The Environmental Research Laboratory in Duluth, Minnesota develops methods,
conducts laboratory and field studies, and extrapolates research findings
—to determine how physical and chemical pollution affects aquatic life
--to assess the effects of ecosystems on pollutants
—to predict effects of pollutants on large lakes through use of models
--to measure bioaccumulation of pollutants in aquatic organisms that are
consumed by other animals, including man.
This report provides a capability to not only predict more accurately the
types of organic materials anticipated from chlorine and ozone disinfection
but also the biological impact (i.e., toxicity and degradability) of these
"second-order" products.
.Donald I. Mount, Ph.D.
Director
Environmental Research Laboratory
Duluth, Minnesota
iii
-------
ABSTRACT
Chlorine has been found to react readily in an aqueous medium with a
variety of materials (a-terpineol, oleic acid, abietic acid, cholesterol,
etc.) known to be present in waste waters subjected to chlorine renovation
processes. The product distribution varies with pH, with a generally higher
level of chlorine incorporation occurring with decreasing pH. The individual
components (chlorides, chlorohydrins, epoxides, etc.) are predominantly those
expected on the basis of the initial ionic attack of a positive chlorine
species. Examples of aromatic compounds that were examined followed the
generally accepted rules for substituent effects such that the introduction
of a chlorine reduced the susceptibility to further reaction and that phenols
were the most vulnerable to electrophilic attack. The aqueous chlorination
of biphenyl produced predominantly the mono- and dichloro species.
Aqueous ozonation of a-terpineol and oleic acid produced olefinic
cleavage products (i.e., aldehydes, ketones, and carboxylic acids) typical
of those found previously in non-aqueous systems.
. In a test to examine the biological effects associated with chlorine
incorporation, the toxicity of a given system to Daphnia magna generally
increased with chlorine content. The excellent correlation of these results
in a Hansch structure-activity analysis with phenols suggests wide applicabi-
lity of this technique for predicting the effects of a given chemical on
the environment. In this correlation the dominant physical parameter was
the partition coefficient, which, in turn, led to the development of a rapid
method for determining the partition coefficient by using the retention
properties of the compound in question on a "reverse-phase" high pressure
liquid chromatography column. In addition, as part of this overall evaluation
of the hydrophilicity of phenols, synthetic procedures were developed for
the preparation of chlorophenylphenols (i.e., the presumed metabolic products
of PCB's).
The effects of chlorination on biological oxygen demand (BOD) were
examined by comparing the BOD requirements of a sample containing a given
parent system vs. that of its chlorinated progeny. The results indicate
that the chlorinated material is generally degraded less than the parent
and that the lowered BOD values appear, at least for phenols, to be associated
with the increased toxicity of the chlorinated material to the degrading
species.
IV
-------
CONTENTS
Foreword iii
Abstract iv
List of Tables vii
Acknowledgments viii
Sections
1. Conclusions 1
2. Recommendations 3
3. Introduction 4
4. Chemical Studies
Aromatic Chlorination 17
a-Terpineol - Aqueous Chlorination 20
a-Terpineol - Aqueous Ozonation 28
Oleic Acid - Aqueous Chlorination 30
Fatty Acids - Aqueous Ozonation 33
Resin Acids - Aqueous Chlorination 38
Cholesterol - Aqueous Chlorination 46
Preparative Methods for Chlorophenyl Phenol Synthesis . . 48
5. Biological Studies
Structure-Toxicity Correlations of Phenolic Compounds
Determined with Daphnia magna 54
Partition Coefficients via High-pressure Liquid
Chromatography 60
Biological Oxygen Demand (BOD) of Chlorinated Effluents . 65
Appendices
A. Summary of Literature on Daphnia magna Toxicity 70
B. Computer Program for Computation of LD5Q 78
-------
FIGURES
Number Page
1
2
3
4
5
6
7
Relationship between the aqueous chlorination products
The chlorination of dehydroabietic acid and abietic acid. .
Log of the percent survivors vs. time, using o-cresol . . .
Probability of survival vs. log of the molar concentration,
Plot of log k1 vs. volume per cent of water for p_-chloro-
Plots of log [l/(k' + 1)] and k' vs. mole per cent of water
for o-chloroohenol
40
49
55
64
64
-------
TABLES
Number Page
1 Percentage Chlorine Uptake by Various Aromatics at Three
pH Values 19
2 Chlorine Incorporation into the Biphenyl Nucleus Under
Various Aqueous Conditions 19
3 Products Formed in the Aqueous Chlorination of a-Terpineol 21
4 Percentage Distribution of Chlorination Products of a-
Terpineol (1) at pH 2.2 and pH 10 22
5 Liquid Chromatographic Separations of a-Terpineol Chlori-
nation Products 24
6 Chlorination Products from Oleic Acid 32
7 Aqueous Ozonation Products of Oleic Acid 34
8 Aqueous Ozonation Products of Linoleic Acid 36
9 Percentage of Compounds Formed by Chlorination of Abietic
Acid at Various pH Values 42
10 Percentage of Compounds Formed by Chlorination of Choles-
terol at Various pH Values 47
11 Toxicity of Substituted Phenols to Daphnia Magna 56
12 Comparison of Observed and Calculated Biological Responses
for Daphnia Magna Toxicity in the Presence of Substituted
Phenols 62
13 Partition Coefficients of Anilines from HPLC Retention
Times 63
14 Partition Coefficients of Phenols from HPLC Retention Times 63
15 Summary of BOD Results for Phenols 66
16 Summary of BOD Results for Benzoic Acids 68
17 Summary of BOD Results of Anilines 68
-------
ACKNOWLEDGMENTS
The authors wish to acknowledge the valuable contributions to the
program that were made by the following individuals:
Research Fellows
Graduate Students
Undergraduates
Technician
Research Chemist,
Lake Superior Basin Studies
Program
Dr. Ted Garbacik
Dr. Richard Hallcher
Dr. Herbert Kopperman
Dr. Gerald Putz
Sr. Agatha Riehl, Ph.D.
Mr. Ned Hillerin
Mr. Robert E. Carlson
Ms. Deborah Massey Jacobson
Ms. Kathleen Mielke
Mr. John Nelson
Mr. Robert Schmitt
Mr. Steve Davis
Mr. Duane Long
We would also like to express our appreciation to Dr. Gary Glass, Dr. Ken-
neth Biesinger and the staff of the Environmental Research Laboratory,
Duluth, for providing the necessary space and advice to obtain a meaning-
ful bioassay. The computer programs, which are an integral part of the
bioassay and structure-activity analysis, were developed with the invaluable
assistance of Dr. Donald Harriss, Department of Chemistry, University of
Minnesota, Duluth.
vm
-------
SECTION 1
CONCLUSIONS
Chlorine is readily incorporated into a variety of organic materials
known to be present in water subjected to chlorine-renovation procedures.
The observed products can be predicted on the basis of commonly used mecha-
nistic considerations. In addition, these second-generation materials
generally have increased toxicity or possess structural features (i.e., an
epoxide or reactive alkyl halide) that have been associated with carcino-
genicity.
The major products formed in the aqueous chlorination of a-terpineol (1_)
apparently can be accounted for by two trans Markovnikov adducts of the
elements of hypochlorous acid. The pH profile of this reaction and the
formation of other minor products can be related to the major trans adducts
by secondary reactions that appear to follow the expected steric, torsional,
and electronic demands of the menthane derivatives. These results will be
useful not only in cataloging toxicity information, but also perhaps in
providing a mechanistic basis for a study of related chlorinated terpenoids.
Cholesterol and oleic acid provide a much less complex mixture of
products; the chlorohydrins predominate in both cases. The isolation of
cholesterol epoxide further illustrates the possibility of obtaining this type
of material during dilute aqueous chlorination.
The resin acids (abietic and dehydroabietic acid) give a variety of
compounds upon chlorination that can be interrelated through various synthe-
tic operations. An important aspect of this portion of the study was the
observation of an apparent "free-radical" chlorination at the benzilic posi-
tion on dehydroabietic acid.
The aqueous ozonation studies confirm that mechanistic considerations
developed in non-aqueous cases can be applied to the prediction of products
from ozone addition to dilute solutions of unsaturated organics in water.
Hansch "structure-activity" relationships are useful in predicting
the potential impact of various organic materials on aquatic organisms.
The dominant feature in the observed toxicity of phenols to Daphnia magna
was the lipophilic nature of the compound as represented by the partition
coefficient. The partition coefficient of a compound has been shown as part
of this overall study to be readily obtained from its retention properties
on a "reverse-phase" HPLC column. This ability to rapidly obtain a measure
of a compound's hydrophilicity has implications for predicting properties
other than toxicity (i.e., bioaccumulation).
-------
The BOD test should not be applied to situations involving chlorinated
organics, because apparent BOD reduction is derived from an enhanced re-
sistance to degradation or increased toxicity to the microbial population
used in the test, or both. In addition, the present method of comparing a
parent compound and its chlorinated progeny appears to be a more sensitive
probe of the relative degradiye potential, as previous studies have not
accounted for the small fraction of chlorinated orgam'cs in comparison with
the unchlorinated organics in the renovated water.
-------
SECTION 2
RECOMMENDATIONS
1. The detailed information on the products obtained from the chlorina-
tion of a-terpineol (or one of the other systems studied) should be applied
to a situation (e.g., a pulp mill effluent) where the possible products can
be most readily observed.
2. The aqueous chemistry of chloramines with very reactive organics
such as phenols should be examined.
3. The cause of an observed BOD reduction, i.e., whether the organics
show a resistance to degradation, should be determined for several different
systems. This could be done by quantitatively examining the chlorinated
organic before and after the test period.
4. The success in rapidly obtaining information on hydrophilicity by
HPLC retention times and the demonstrated importance of this parameter in
predicting toxicity should be applied to other areas. For example, there
should be a gross HPLC analysis of complex effluents to determine which
fraction would have the maximum probability of exhibiting toxicity or bio-
accumulation effects.
5. Other methods of wastewater renovation (in particular, with ozone
and ClOg) should be examined chemically and biologically to ascertain if these
methods represent a suitable alternative to chlorination.
6. The chemical and biological implications of applying chemical
disinfectants to waters containing polynuclear aromatic hydrocarbons (PAH)
should be investigated.
-------
SECTION 3
INTRODUCTION
Chi on" nation is the predominant technique used for water renovation and
disinfection. The process has been applied to wastewater treatment problems,
to the disinfection of drinking water and the preservation of acceptable
water quality through distribution systems, to the solubilization of sludge
(a "superchlorination" process involving large doses of chlorine), to the
maintenance of hygienic conditions in closed swimming areas, and to the
reduction of algal and bacterial growth in cooling towers J~5 The develop-
ment of the technology for the effective application of chlorine has been
considered largely responsible for saving thousands of lives that could have
been lost through contracting any of several possible water-borne diseases.
In short, chlorination has developed into what has been referred to as
"the most valuable and versatile tool available to the water chemist.11'
The possible reaction of chlorine with materials present in the treated
water has long been recognized,^ mainly because of the very practical neces-
sity for using more chlorine than was anticipated to meet given standards
of 'turbidity, BOD, 7 or fecal coliform. Environmentally, chlorine and chlora-
mines (as reaction products of ammonia, ami no acids, or other amines with
chlorine) are considered deleterious,5'8 and considerable effort has been
directed toward their removal by such processes as reduction (e.g., SOg)
or by adsorption-decomposition (activated charcoal), with the result that
documented examples of incorporation of carbon-bound chlorine under con-
ditions used in water treatment have been quite limited. Early chemical
investigations were only initiated when the chlorination process generated
problems of taste and odor.""''
The addition of chlorine to water results in the rapid hydrolysis to
hypochlorous and hydrochloric acids. Moreover, considering the k? of hypo-
chlorous acid, the active chlorine species will be essentially all hypo-
chlorous acid or all hypochlorite in changing from a pH of 5 to a pH of 9J»5
C12 + H2O^HOC1 + H+ + CT
k] = 3.94 x lO'4 at 20-25°
HOC1^±H+ + 9OC1
k2 = 3.2 x ID'8 at 20-25°
In the presence of ammonia a very rapid reaction occurs to generate
chloramine. The subsequent conversion to di- and trichloramine occurs at
a slower rate. This facile generation of chloramine thereby represents
an effective competitive process to the formation of "second-order" chlor
organics where a water sample contains substantial amounts of ammonia. 5
-------
NH3 + HOC1^=± NH2C1 + H20
k3 = 3.6 x 109
Recent determination of the organic content of water samples that are
typically subjected to aqueous disinfection processes has shown that many
compound types are present that could incorporate carbon-bound chlorine.'2-14
These types include aromatic compounds (i.e., phenols, amines, etc.), hetero-
cycles, terpenes, sterols, resin acids, and unsaturated fatty acids. These
studies not only indicate the complexity of the trace organics present in
water, but, coupled with some fundamental organic chemical principles, give
some insight into the possible "second-order" organic material that can be
anticipated from any further chemical transformations. The presence of
substantial amounts of "second-order" chlorinated pollutants arising from
water-renovation processes that are not subject to reductive "dechlorination"
has only recently gained attention because of the isolation of chlorinated
materials from supplies of potable water.6 Of equal scientific significance
is the recent work by Jolley in which a combination of radioactive tracer
techniques and high pressure ion-exchange chromatography demonstrated the
presence of over 50 "new"organochlorine species from a wastewater chlorination
processJ5 The magnitude of the observed 1% chlorine uptake as carbon-bound
chlorine observed by Jolley can best be illustrated by a specific example.
Thus, at an average flow of 43.6 MGD of municipal and industrial waste into
the Western Lake Superior Sanitary District (WLSSD, Duluth, Minnesota,
100,000 population) and the use of 28 Ibs. of C12/MG, the waste-treatment
facility will discharge 21,900 Ibs. of chlororganics into Lake Superior
during the course of a single year. This figure of nearly 11 tons of
yearly discharge is conservative in that average flows were considered and
Jolley was only examining "soluble" organic material, thereby eliminating
from consideration the various types of polymeric material (humic acids,
polypeptides, etc.) and volatiles (chloroform, etc.).
Average flow of municipal and industrial waste
(WLSSD) =43.6 MGD
28 Ibs of C12/MGD = 1,222 Ibs. Cl2/day
1% chlorine incorporated = 12 Ibs.
Average molecular weight of "new" chloror-
ganic = 175 = 60 Ibs chlororganics
produced/day
Yearly discharge (X 365) = 21,900 Ibs./year
An a priori consideration of mechanistic organic chlorine chemistry
suggests that the anticipated chlorine-containing organic products will be
derived either from the attack of an electrophilic chlorine species (often
represented by Cl+) or an aromatic,'7 an olefinic, or other nucleophilic
portion of the molecule or by a free radical process.^ The former process
will generate products such as halogenated aromatic systems (electrophilic
aromatic substitution) and olefin-derived compounds such as chlorohydrins
(addition reactions) and will proceed at a rate proportional to the avail-
ability of the electrons in the substrate. The radical process, although
less likely in the polar aqueous medium, will be most readily observed from
-------
those compounds that can provide a stable radical intermediate (e.g., benzilic
halogenation).
"C1+"
Electrophilic
Substitution
Addition of HOC!
Cl OH
Free radical
Aqueous electrophilic aromatic substitution processes have been thor-
oughly documented for phenols where chlorine can readily be incorporated
throughout a wide range of pH values because of the reactive nature of the
phenolic ring system. In the case of phenol itself, chlorination proceeds
through qrtho and para-chlorophenol to the higher di- and tri-chlorinated
isomers (2,4- and 2,6-dichlorophenol and 2,4,6-trichlorophenol9~^), and
the rate of chlorine incorporation decreases with increasing chlorine con-
tent. Further reactions consist of oxidation to the chloro-orthoquinone
and subsequent cleavage rather than incorporation of additional chlorine
atoms.6
OH OH OH
Cl
Subsequent
cleavage
products
-------
In strongly alkaline media, hypochlorite also results in degradation of
the phenolic ring system. The products are of various types, including
a series of interesting ring-contracted materials (e.g., cyclopentane-
carboxylic acid derivatives) related to some known antibiotic systems.^
Cresols have been found to provide stable chloro-o-benzoquinones. This
type of product has been Sbown to be a component of a Fleached Kraft mill
effluent toxic to salmon.20,21
Cl,
H20
The delignification in pulp bleaching represents an additional example
of phenolic chlorination chemistry. The incorporation of chlorine into the
aromatic nucleus occurs not only by direct introduction, but also by an
alternate process that can be referred to as electrophilic replacement.22
Other phenolic materials would be expected to react in a similar fashion to
the examples cited.
:\
CHOR
C12/H20
Cl
OH
R = H or alkyl
Tetrachloroguaicol
Polynuclear aromatic hydrocarbons (PAH) represent another ubiquitous
compound type that has been shown to incorporate chlorine into the aromatic
nucleus. For example, in a recent study of the aqueous chlorination of the
water-soluble portion of diesel fuel a number of chloronaphthalenes were
observed.23
-------
An olefin represents an additional example of a site subject to electro-
philic attack. In the case of the isolated olefin without hetero atom sub-
stituents the major products of aqueous chlorination are chlorohydrins,
although 1,2-dichloro compounds, epoxides, and other compounds are also
possible. Historically, although some of the oldest known addition studies
dealt with electrophilic additions to monocyclic terpenes,^ aqueous chlori-
nations often produced prohibitively complex mixtures that were unsuitable
for any product or mechanistic study." other olefinic compounds known to
be present in water include unsaturated fatty acids (oleic, linolenic, etc.),
cholesterol, resin acids-.steroids, and other miscellaneous natural and
industrial compounds. The o-halogenation of carbonyl systems also
belongs to this type of reaction when an enol is considered as a reactive
intermediate.
HOC! v C— C and related products
OH Cl
Chlorohydrin
y
\>
_~ c-
-- C - C
\ ^ /
H
Enol
Aromatic heterocycles (i.e., purines and pyrimidines) that are derived
from the breakdown of nucleic acids have been observed by Jo!ley to incor-
porate chlorine. In that study 5-chlorouracil, 8-chlorocaffeine, 5-chloro-
uridine, and 8-chloroguanine and 8-chloroxanthine were present.
The halogenation products of carbonyl compounds vary with pH, but in
the case of the haloform reaction (basic conditions, methyl ketones) the
initial halogenation represents the slowest step. Potential enolic systems
can be found in compounds possessing a carbonyl or functional group (e.g.,
-OH, -NH£) that could represent a carbonyl precursor.
The ultimate products from chlorination of a methyl ketone will be
chloroform and the carboxylic acid salt. Although it is generally considered
that only methyl ketones can ultimately generate haloforms, there is con-
siderable evidence that humic material is the source of the chloroform
derived from chlorination of drinking water^ and that a methyl ketone
equivalent such as a $-keto acid, a s-keto aldehyde, or a e-diketone might
represent the requisite reactive methyl ketone equivalent.
-------
? C12/90H S Faster R fi
R-C-CHo »• R-C-CH?C1 -} ->• R-C-CClo—» R-CV + HCClo
d Slow * d be 3
0 00
II »| H
Methyl ketone equivalents: R-C-CHo-COo.H R-C-CHo-C-R
f f
It is apparent that stable chlororganics may be introduced into aquatic
systems in a host of ways. Adding to the overall concern about the environ-
mental implications of such a phenomenon is the ever-increasing amount of
recycled water that is being used and the possibility that these "second-
order" organo-chlorine molecules might have some of the negative properties
of their "primary" pollutant counterparts.*b,27 jne possible environmental
hazards associated with aqueous chlorination therefore strongly suggest
that viable alternatives should be sought and evaluated.
Ozone represents one potential alternative to the use of chlorine in
water-quality control in spite of the additional cost and the inability to
maintain a residual. These deficiencies in the character of ozone are
frequently offset by its reported superior qualities in removing taste, odor,
and color and by its outstanding disinfecting capability.28
Ozone is a very reactive species with an oxidation potential second only
to fluorine. Ozone is diamagnetic and, unlike oxygen, one should expect the
initial attack of ozone to be non-free-radical, with ozone acting as an
electrophile or as a 1,3-dipole, or both.
..©©..
* / V "~* A n " A "
•0, ;0: -*0. 0'® -0-, 0'
Information is limited, however, on the aqueous chemistry of ozonation,
although kinetic studies have shown that the mechanism of ozonation apparent-
ly does not vary with solvent polarity.29,30 Fortunately, therefore, much
of the initial work already done with organic solvents can be applied to
aqueous systems.
Ozone decomposes in water, and the sole decomposition product is
oxygen. Although the mechanism of decomposition is not agreed upon,25,26
the rate of this reaction increases with an increase in pH or an increase
in salt concentration.27 Most of the proposed ozone decomposition mecha-
nisms are free radical25,26, but the rate of aqueous decomposition is slow
-------
with respect to ozonolysis^ and for this reason the free-radical contribu-
tion by the decomposition intermediates is generally ignored.
"Among the organic groupings which can be oxidized by ozone are ole-
finic and acetylenic carbon-carbon multiple bonds; aromatic, carbocyclic
and heterocyclic molecules; carbon-nitrogen and similar unsaturated groupings;
nucleophilic molecules such as amines, sulfides, sulfoxides, phosphines,
phosphites, arsines, selenides, etc.; carbon-hydrogen bonds in alcohols,
ethers, aldehydes, amines, hydrocarbons, etc.; silicon-carbon, silicon-
silicon. and silicon-hydrogen bonds; and carbon-metal bonds of various
types. "30
N — ; :NR3; R2S: ; R2S= ; :PR3; :P(OR)3;
C
0
:AsRa; e: '• H-C^as in R3C-H, R-C-H, R2N-CR2
R-S-CR2, R-0-CR2, etc.; ^Si-
H
1 A
C-Mg— , —C-Hg- , etc.
"Carbon-carbon double bonds are usually the most reactive in the above
systems toward ozone, but nucleophiles such as arsines and selenides, as
well as certain carbon-nitrogen double bonds, are nearly as reactive, and,
in some cases, are more reactive. Carbon-hydrogen and silicon-hydrogen, etc.,
bonds are usually the least reactive of the preceeding groupings present in
the material being ozonized."30
Alkenes represent the most reactive of a number of organic systems
that are vulnerable to ozone attack. Criegee has proposed a three-step
mechanism which explains the major products that are formed from alkene
ozonolysis, and although other mechanisms have been proposed,3" a recent and
more comprehensive version of his original proposal is now generally ac-
cepted. 31 -33
"In aqueous media, the peroxidic ozonolysis product should be a hydroxy
hydroperoxide. Although only a few studies of ozonolysis have been made
in water media 34-38t the results indicate that the hydroxy hydroperoxide
is fairly easily decomposed to an aldehyde (or ketone) or to a carboxylic
acid, as shown below, although heat may sometimes be necessary to effect
the transformation. Since aldehydes can also be converted to carboxylic
acids by ozone (see later discussion) the end products of these reactions
would be further oxidized by ozone only slowly and with great difficulty."30
10
-------
Revised Criegee Ozonolysis Mechanism
-'0
R2C=CR2 R2C— CR2
R R \ --0+ .. /O 0.
C-04-^ - I + R2C=0. - ^ / \
* '
R R
0-0 vO-O-H
\ / \ p r X
C-O-O-J— R?C /CR? K2L\ X = OH, -OCH3,
y x %
cr~° -OCCH3, etc.
"With certain olefins, having two bulky substitutents at the carbon, or
three at both carbons of the double bond, oxide formation competes with
ozonolysis. These reactions appear to involve a purely electrophilic
ozone attack, followed by loss of molecular oxygen, and occur both in polar
(e.g., water) and non-polar solvents."30
R. xO-O-H -H202 R\
_
c — 0 (aldehyde or ketone)
Rfi U
\j~ n
(H)
Hydroxyhydroperoxide
"N / °"H
c "HOH >_ R-fc-OH (carboxylic acid)
H 0-0-H
11
-------
"Acetylenic compounds undergo ozonolysis at the triple bond. The Crie-
gee mechanism appears to apply, although few mechanistic studies have been
made. Based on this mechanism as shown below, the peroxidic intermediate
should be that shown, and the final product should be a carboxylic acid."30
R—C = C—R
A
0 p-O-H
|I /f ' I
R-C— C—R
-H
0-H
R-C— 0 — C — R
0-H
2 R-C.-0-H
0
"The ozonation of aromatic compounds appears to involve both ozonolysis,
at the most reactive aromatic bond, and electrophilic ozone attack at indivi-
dual carbon atoms.3^ In regard to ease of attack, the unsubstituted benzene
ring is much less reactive toward ozone than in an olefinic double bond."30,39
The effect of substituents on the rate of ozonolysis is similar to other
electrophilic additions where alkyl, aryl, oxygen, etc. substituents facili-
tate the reaction, whereas groups such as nitro, carboxyl, halogen, and
sulfonic acid substituents slow the reaction. The cleavage products of ben-
0 ,0
zene ring systems are the expected glyoxals
0
(R-C-C02H).
(R
-C-C-H) and glyoxalic acids
The only aromatic system that has been studied to any extent in a-
queous media is phenol and its homologs. A study of ozonation of phenol
itself suggests the intermediacy of several intermediates including catechol ,
the orthoquinone, and muconic
Other organic compound types would also be expected to react with ozone.
Polynuclear aromatics such as anthracene, naphthalene, and phenanthrene
are more reactive than benzene derivatives, but less reactive than olefins.
Amines, sul fides, and selenides are oxidized to their corresponding oxides,
12
-------
Electrophilic
Attack
Phenol
1,3-Dipolar
cycloaddition
OH
Products from
further
ozonolyses
Orthoquinone
Muconic acid 1 1
and in some cases ozonation is faster than with double bonds. Carbon-hydrogen
bonds can also be oxidized by ozone through the hydrotrioxide intermediate.^
no n
:3_^ C6H5C-0-0-0-H _ > C6H5C02H
-02
H H
CH3 CH3
CH3 CH3
Stable hydroperoxide derivatives can be formed during the ozonation pro-
cess as illustrated by Criegee's study of the ozonolysis of a cyclic olefin
in the presence of ammonia^ and in the ozonolysis of a cyclic sulfone.29
13
-------
REFERENCES
1. White, G. C., "Handbook of Chlorination," Van Nostrand-Reinhold Co.,
New York, N.Y., 1972.
2. American Water Works Association, "Water Quality and Treatment, McGraw-
Hill Book Co., New York, N. Y., 1971.
3. McCoy, J. W., "The Chemical Treatment of Cooling Water," Chemical Pub-
lishing Co., New York, N. Y., 1974.
4. James, G. V., "Water Treatment," CRC Press, Cleveland, Ohio, 1971.
5. Johnson, J. Donald, "Disinfection - Water and Wastewater," Ann Arbor
Science Publishers, Ann Arbor, Michigan, 1975.
6. Morris, J. C., "Formation of Halogenated Organics by the Chlorination
. of Water Supplies, U. S. Environmental Protection Agency, Washington,
D. C., Publication EPA-600/1-75-002, 1975.
7. Barnhard, E. L., and G. R. Campbell , "The Effect of Chlorination on
Selected Organic Chemicals," U. S. Environmental Protection Agency,
Washington, D. C., Publication EPA-12020EXG03/72, 1972.
8. Brungs, W. A., J. Water Poll. Contr. Fed.. 4^, 2180 (1973).
9. Burttschell, R. H., A. A. Rosen, F. M. Meddleton, and Morris B. Ettin-
ger, J.Amer. Water Works Assoc., 51, 205 (1959).
10. Howard, N. C. and R. E. Thompson, New England Water Works Assoc., 40,
276 (1926). ~
11. Lee, G. F. and J. C. Morris, Int. J. Air Wat. Poll.. 6_, 419 (1962);
J. C. Vaughn, Sci. Techno!. 1, 703 (1967).~
12. Keith, L. H., "Identification and Analysis of Organic Pollutants in
Water," Ann Arbor Science Publishers, Ann Arbor, Michigan, 1976.
13. Pitt, W. Wilson, Jr., R. L. Jolley, and C. D. Scott, Environ. Sci.
Techno!.. |, 1068 (1975).
14. Keith, L. H., Environ. Sci. Techno!., ]£, 555 (1976).
15. Jolley, R. L., "Chlorination Effects on Organic Constituents in Effluents
14
-------
from Domestic Sanitary Sewage Treatment Plants," Oak Ridge Nat. Labora-
tory, Oak Ridge, Tennessee, Publication ORNA-TM-4290, 1973.
16. Consoer, Townsend, and Associates, "Wastewater Treatment Facilities
Design-Data for Preparation of Contract Drawing," Consulting Engineers
for the Western Lake Superior Sanitary District, Duluth, MM, 1972.
17. De LaMare, P. B. D. and J. H. Ridd, "Aromatic Substitution Nitration
and Halogenation," Academic Press, New York, N. Y., 1959.
18. Morrison, R. T. and R. N. Boyd, "Organic Chemistry," Allyn and Bacon,
Boston, Mass., 3rd Ed., 1973.
19. Moye, C. J. and S. Sternhell, Aust. J. Chem.. TJ., 2107 (1966). See
also: W. B. Turner, "Fungal Metabolites," Acattemic Press, New York,
N. Y., 1971, pp. 125-127.
20. Das, B. S., S. G. Reid, J. L. Betts and K. Patrick, J. Fish. Res. Bd.
Can., H, 3055 (1969).
21. Gess, J. M. and C. W. Dence, Tappi, J4f 1114 (1971).
22. Sarkanen, K. V., Pure and Applied Chem.. £, 219 (1962).
23. Reinhard, M., V. Drevenkar and W. Giger, J. Chromatgr., 116, 43 (1976).
24. Simonsen, J. L., "The Terpenes," Vol. 1, Part II, Cambridge University
Press, Cambridge, England (1953).
25. a) Slawinski, K., Chemik Pol ski. 1_5, 97 (1917) and b) Slawinski, K. and
G. Wagner, Chem. Ber., 32^ 2064 (T899).
26. Brooks, G. T., "Chlorinated Insecticides, Volumes I and II, CRC Press,
Cleveland, Ohio, 1974.
27. Hutzinger, 0., S. Safe, and V. Zitko, "The Chemistry of PCB's," CRC
Press, Cleveland, Ohio, 1974.
28. Evans, F. L., "Ozone in Water and Wastewater Treatment," Ann Arbor
Science Publishers, Ann Arbor, Michigan, 1972.
29. Bailey, P. S., J. W. Ward, R. E. Hornish, and F. E. Potts III, "Advances
in Chemistry Series," Am. Chem. Soc., Washington, D. C., Publication
No. 12, 1972, p. 1.
30. Bailey, P. S. in "Proceedings of the First International Symposium
on Ozone," International Ozone Institue, Waterbury, Conn., 1975
p. 101.
31. Davison, R. and C. Hewes, Amer. Inst. Chem. Engin. J., 17, 141 (1971).
15
-------
32. Kilpatrick, M. and C. Herrick, J. Amer. Chem. Soc., 78_, 1784, 6423,
(1956). —
33. Cornelia, C., J. Amer. Water Works Assoc., 64_, 39 (1972).
34. Pryde, E. H., D. J. Moore, and J. C. Cowan, J. Am. Oil Chemists Soc.,
45, 888 (1968).
35. Criegee, R. and G. Lohaus, Ann. Chem.. 583, 1 (1953).
36. Fremery, M. I. and E. K. Fields, J. Org. Chem., 28_, 2537 (1963).
37. Fields, E. K., "Advances in Chemistry Series," Amer. Chem. Soc.,
Washington, D. C., Publication No. 51, 1965, p. 99.
38. Sturrock, M. G., E. L. Chine, and K. R. Robinson, J. Orq. Chem., 28_,
2340 (1963). —
39. Bailey, P. S., Chem. Rev.. 5J., 925 (1958).
40. Fermery, M. and E. Field, J. Org. Chem.. 29_, 2240 (1964).
16
-------
SECTION 4
CHEMICAL STUDIES
AROMATIC CHLORINATION
Results and Discussion^
The introduction of chlorine into an aromatic system represents an
example of electrophilic aromatic substitution, for which there is a single
unifying mechanism with respect to substrate.*»3»4
When the aromatic system is monosubstituted, the reaction may be faster
or slower than with benzene itself, and the aromatic electrophilic substi-
tution process will result in a product that is ortho (1,2-disubstitution),
meta (1,3-disubstituted), or para (1,4-disubstituted).5
u
Activating groups are those which increase the reaction rate, whereas
deactivating groups are those which decrease the reaction rate. All groups
that give predominantly meta products are deactivating; most of the ortho-
para-directing substituents are activating. The notable exceptions are
the halogens. Although a halogen is deactivating (i.e., less reactive than
without the halogen) the products of subsequent electrophilic substitution
will be predominantly ortho-para.
Monosubstituted aromatics were exposed to low concentrations (7.0 x
10~4 M) chlorine for twenty minutes (Table 1). The extent of chlorine
incorporation followed well-recognized trends, i.e., aromatics containing
"activating" groups such as hydroxyl, ether, amine derivatives, or alky]
underwent electrophilic aromatic substitution faster than those containing
"deactivating" groups such as nitro, chloro, nitrile and carbonyl. Phenol
is unique in its ready chlorination at high pH values; although the chlorina-
ting agent is hypochlorite, the substrate is the very reactive phenolate
anion.
-------
The aqueous chlorination process was analyzed in detail by using bi-
phenyl as the aromatic species. Biphenyl was chosen for study because the
methodology for isomer recognition is available and because considerable
interest has recently centered on the possible production of polychlorinated
biphenyls (PCBs) at waste-treatment facilities known to receive biphenyl.6
The results shown in Table 2 confirm the possibility of chlorine incorpora-
tion into the biphenyl nucleus under various aqueous conditions. The
extent of chlorobiphenyl production was dependent on pH. Above pH 4
extended reaction times were required for extensive chlorine incorporation.
Substantial amounts of higher chlorinated isomers occur at increased chlorine
concentrations, an important observation if potential problems associated
with "superchlorination" for such purposes as sludge solubilization are being
considered.
Experimental
Procedure for Aromatics Study (Table 1) —
All reactions were carried out in a 500-ml volumetric flask. To each
flask was added 12.72 ml of the 3.84-mg/ml NaOCl solution, HC1 (for pH
adjustment), and the test solution. The reaction temperature was maintained
at 25° ± 0.2°C by using a constant temperature bath. A 20-ml aliquot was
taken at twenty minutes and added immediately to a flask containing 15 ml
of 0.00543N N32S203 solution, 5 ml of HOAc, 2 ml of KI (50% aqueous) and
1-2 ml of freshly prepared starch solution. This was titrated to a faint
purple color with 0.096 mg/ml of NaOCl solution.
Procedure for Biphenyl Study (Table 2) --
To five of the bottles containing 100 ml of distilled water and saturated
biphenyl, 100 ppm of Ca(OCl)2 were added. The remaining five bottles,
containing water and biphenyl only, served as controls. The pH of the final
solutions was approximately 5.5. Samples were extracted at intervals of one
day, one from each concentration of Ca(OCl)2 and a control, resulting in
exposure periods of 1, 2, 3, 4, and 5 days. The entire contents of a bottle
were transferred to a 250-ml separatory funnel and extracted three times
with 25 ml of hexane. The bottles were rinsed with the first 25-ml aliquot.
The combined hexane extracts were filtered through anhydrous sodium sulfate
and concentrated in a Kunderna-Danish evaporator concentrator. The hexane
concentrates were analyzed with electron capture gas chromatography (EC/GC)
under the following conditions.
Analysis conditions (samples 1-9) -- Instrument: Hewlett-Packard 5753A
(Ni-63 Electron Capture Detector).Column: 2M x 1/4" glass-4% XE-60 sili-
cone on 80/100 mesh HP Chromosorb W. Injection temperature: 200°C. Column
temperature: 130°C isothermal. Detector temperature: 250°C. Carrier:
helium @ 60 ml/min. Purge: 10% methane/argon @ 120 ml/min. Pulse interval:
50 ysec.
Analysis conditions (samples 10-15) — Instrument: Tracer Model 550
Gas Chromatograph (tritium-electron capture detector). Column: 6' x 4mm
ID glass column — 3% Ov - 7 on 80/100 HP chromosorb W. Injection tempera-
ture: 200°C. Carrier: nitrogen @ 70 ml/min. Purge: nitrogen @ 30 ml/min.
18
-------
TABLE 1. PERCENTAGE CHLORINE UPTAKE BY VARIOUS AROMATICS AT THREE pH VALUES
pH
(9.5 ± 0.6 X W M)
Phenol
Anisole
Acetanihde
Toluene
Benzyl alcohol
Benzonitrile
Nitrobenzene
Chlorobenzene
Methyl benzoate
Benzene
Chlorine (7.0 X 10~4;'
3
% Cl (uptake)
97.8 ± 0.1
80.7 ± 0.2
55.3 ± 0.5
11.1 ± 0.1
2.3 ± 0.2
2.1 i 0.2
1.8 ± 0.1
1.8 ± 0.1
1.8 ± 0.2
1.5 ± 0.1
I/), 20 min, 25°C.
7
%CI
97.6 i 0.1
11.4 ± 0.4
3.4 ± 0.2
2.9 ± 0.4
—
—
—
—
—
—
10.1
%CI
97.6 ± 0.2
2.8 ± 0.3
—
—
—
—
—
—
—
—
TABLE 2. CHLORINE INCORPORATION INTO THE BIPHENYL NUCLEUS
UNDER VARIOUS AQUEOUS CONDITIONS
Chlorine
source
Ca(OCI)2
Ca(OCl)2
Ca(OCI)2
Ca(OCI)2
Ca(OCI)2
Ca(OCI)2
Ca(OCI)2
Ca(OCI)2
Ca(OCI)2
Cl,
NaOCI
ci,
NaOCI
Cl,«
NaOCI
pH
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
5.5
2.1
2.8
4.9
10.4
2.2
7.0
Chlorine,
ppm
100
100
100
100
10
20
35
50
100
10.9
295
266
830
1350
2950
Reaction
time, hr
24
48
72
120
120
120
120
120
120
0.25
0.25
0.25
0.25
0.25
0.25
Reaction,
%
0.4
1.0
1.7
2.5
0.004
0.02
0.04
0.15
0.27
2.2
9.3
0.1
0.6
16
5.2
2-
15
32
56
69
0.1
0.44
1.4
4.0
8.8
120
450
5-10
40
180
280
3-,4-
10
27
47
82
0.12
0.51
1.2
4.9
7.4
10
190
80
no
2,2' 2,3'-2,4' 4,4'-
20
10 130 40
80 370 500
80 30
Saturate
o Higher
d biphenyl solution found experimentally to be 6.0 mg/l.
chlorinated isomers also present.
19
-------
Compound identification and calibration -- Chlorinated isomers were
designated by comparison with pure materials (as determined by GC-MS).
The percentage reaction is based on peak areas as compared to standards
and on the experimentally determined solubility of biphenyl (6.0 mg/1).
REFERENCES
1. Carlson, R. M., R. E. Carlson, H. L. Kopperman, and R. Caple, Environ.
Sci. Techno!.. |, 674 (1975).
2. Norman, R. 0. C., et al., "Electrophilic Substitution in Benzenoid
Compounds," American Elsevier, New York, N. Y., 1965.
3. de la Mare, P. B. D. and J. H. Ridd, "Aromatic Substitution - Nitration
and Halogenation," Academic Press, New York, N. Y., 1959.
4. Cohen, S. G., A. Streitwieser, Jr., R. W. Taft in E. Berliner, ed.,
"Progress in Physical Organic Chemistry - Vol. 2," Interscience,
New York, N. Y., 1964.
5. Ferguson, L. S., Chem. Rev., 50_, 47 (1952).
6. . Gaffney, D. E., Science, 183, 367 (1974).
a-TERPINEOL (CHLORINATION)
Results and Discussion
Historically, some of the oldest known addition studies dealt with
electrophilic additions of halogens to monocylcic terpenes.' Not sur-
prisinqly, the reactions were somewhat restricted to conditions that pro-
duced manageable results (i.e., bromine, in a non-polar solvent). Furthermore,
aqueous halogenations, especially aqueous chlorinations, often produced pro-
hibitively complex mixtures of second-order chlororganics that were un-
suitable for any subsequent product or mechanistic studies. However, the
possibility of forming stable and toxic chlorinated derivatives from the
widespread use of chlorine in disinfection, wood-pulp bleaching, and related
processes2»3»4 mac|e imperative an examination of the aqueous chlorination
chemistry of a common monocyclic terpene.
We have, therefore, interpreted in detail the aqueous chlorination
chemistry of a-terpineol (£-menth-l-en-8-ol, ]_), an excellent example of
ubiquitous naturally occurring monocyclic terpene.5-9 Although an earlier
investigation of the interaction of a-terpineol (1_) with sodium hypochlorite
gave an unidentified mixture of products and was inconclusive,'0''' we
reapproached this problem with the aid of modern chromatographic techniques.
Our ultimate goal was to provide a mechanistic interpretation'2 and a pH
profile of the observed product distribution to aid in the prediction of
products derived from related systems.
20
-------
The aqueous chlorination of a-terpineol (1_) was, therefore, examined
over a pH range of about 2 to 10, the lower pH corresponding closely to a
saturated chlorine solution and the higher pH to a sodium hypochlorite
solution. Considerable effort was made to detect and identify every obser-
vable compound, as relative yields have less significance when one is con-
cerned with bioaccumulation and toxicity. In the present analysis (LC and
glc) products in the half-percent range could be reproducibly detected
(Table 3).
TABLE 3. PRODUCTS FORMED IN THE AQUEOUS CHLORINATION OF a-TERPINEOLa
OHO>
Dichloro Derivatives"
Cl
ft -2,a -6-dichloro-ds
p -menthane-1,8-diol (2)'
OH
1,2-dichloro-p-
menthane-8-ols (3)c
Monochloro Derivatives"
OH
Non-chlorinatedh Products
O.
a-2-chloro-cis-p-
menthane-1,8-diol (4)'
OH
OH
j8 -2-chloro-f rans -p -
menthane-1,8-diol (5V
p -menthane-a-1,2-epoxy-
8-ol(7)'
OH
p-menthane-£i-l,2-epoxy-
8-ol (8V
OH
OH
6-chloro-cii -p -menthane-
1,2,8-triol (6V
OH
2-endo -cineolylol (9)'
"Not all of the products are formed at any given pH; see product distribution as function of pH in Table 2.
h Chlorine content established by mass spectral analysis and microanalysis.
'Acceptable names based on alternate p-menthane nomenclature are, for 4, 1,8-dihydroxy-neoisocarvomenthyl
chloride; for S, 1.8-dihydroxyneocarvomenthyl chloride; for 7. ?ranj-p-methane-l,2-epoxy-8-ol; for 8, cis-p-
menthane- l,2-epoxy-8-ol.
The distribution of these products as a function of pH is given in
Table 4. The distribution was similar from pH 2 to 6, and, therefore,
only one set of values (at pH 2.2) is given for this range. Likewise, the
results above pH 7.9 were similar; only the values for pH 10 are provided.
The results are reproducible for a given set of experimental conditions,
and the percentages represent all observable products. The percentages
21
-------
agree with actual isolated yields, and apparently all compounds present at
0.5% or higher were detectable by analytical HPLC.
The chlorine addition reactions, whether starting with chlorine gas
(for highly acidic reactions) or sodium hypochlorite solutions, were run
with about two equivalents of available chlorine. This quantity is essen-
tially the minimal quantity necessary to insure the complete reaction of
a-terpineol (1_). Reactions with a larger excess of chlorine, and reaction
times beyond the disappearance of a-terpineol (1_), led to only a very small
and slow increase in the chlorine content of the chlorinated mixture,
presumably via a free radical substitution or a dehydration and addition
sequence, or both. Thus, the product distribution was quite insensitive
to small changes in reaction conditions.
TABLE 4. PERCENTAGE DISTRIBUTION OF CHLORINATION PRODUCTS OF a-TERPINEOL (1)
_ AT pH 2.2 and pH
Compoundc pH 2.2 pH 10
1
2_
3_
4_
5
6^
7_
8
i
0
9
2
29
53
4
1
1
1
0
0
0
42
0
19
39
0
0
a A LC analysis on a Corasil column of the ether extracts.
b The data are reproducible for a given set of experimental conditions
and are believed to be accurate to ±1%. The percentages were ob-
tained by integration of results on LC column and are not corrected
for molar purposes.
c Toxicity: With the exception of 3_, the compounds formed have a
toxicity to Daphm'a magna, LCso (48 hrs.), similar to or even less
than that of a-terpineol (120 ppm). The dichlorides 3_ are an order
of magnitude more toxic at about 15 ppm.
An examination of the major products formed at a pH of 2.2 and 10
revealed a self-consistent pattern. The major product under the acidic
conditions was assigned to the trans-diaxial adduct 5_ of hypochlorous acid.
The configuration at £% 1S readily established by pmr, & \\2 = 4.09, J2e,3e "
22
-------
the assignment at C] is based on mechanistic grounds
2e 3a = - z> u e assgnmen a ] s as
ntr conversion into the corresponding epoxide 7_.
Not unexpectedly, the major products under basic reaction conditions
were the a-epoxide _7_ derived from the cyclization of the diaxial chloro-
hydrin 5^ and the trans-diequatorial chlorohydrin £.
The major products formed in the aqueous chlorination of a-terpineol (1_)
can, therefore, be accounted for by two trans Markovnikov adducts of the
elements of hypochlorous acid. The pH profile of this reaction and the
formation of other minor products can be related to the major trans adducts
by secondary reactions that appear to follow the expected steric, torsional ,
and electronic demands of the menthane derivatives. These interactions are
summarized in the flow diagram in Figure 1_. These results will be useful
not only in cataloging toxicity information, but will provide a mechanistic
basis for a study of related chlorinated terpenoids.
acidic
ocr/Hoci
basic
dichlorides chlorohydrins
3 and 5
6-epoxide a-epoxide dichloro diol
i Z. "L
I " I
bicyclic ether
9
chloro triol
6
a-epoxide
7_
chloro triol
i
chlorohydrin
4
Figure 1. Relationship between the aqueous chlorination products of a-
terpineol (1_).
Experimental
Measurements of pH were recorded by a Corning Scientific Instruments
Model 7 pH meter. Analytical vapor phase chromatography was performed on
23
-------
a Tracer Model 550 (FID) detector). Preparative work was carried out on
a Varian Autoprep Model A-700 (TC detector). Melting points were determined
with a Thomas Hoover capillary melting point apparatus and are uncorrected.
Elemental analyses were performed by Schwarzkopf Microanalytical Laboratory,
Woodside, New York. Nmr spectra were obtained with a Varian Associates
Model A-60D Spectrometer; tetramethylsilane was used as an internal reference.
Mass spectra were obtained on a Varian CH6 instrument and infrared on a
Beckman IR 33. The liquid chromatographic monitoring of samples was per-
formed on 1/8 x 2' Corasil II columns that were mounted in a Waters Associates
ALC 202 (differential refractive index detector). Product resolution was
accomplished in two solvent systems and their separations are summarized
in Table 5.
TABLE 5. LIQUID CHROMATOGRAPHIC SEPARATIONS OF a-TERPINEOL CHLORINATION
PRODUCTS
Polar
Non-Polar
Solvent System:9 99% CHC13, 1% CHaOH Solvent System:b 50%
Compound Elution Time
(min)
2_ 1.25
4 1.75
5. 2.25
i 6.5
Compound
3_
1
7.
£
8_
Elution Time
(min)
2.0
2.5
5
6
7
a Corasil II column, 0.5 ml/min
D Corasil II column, 2.0 ml/min
Reagents --
The a-terpineol (]_) used was obtained from Eastman Organic Chemicals
and was purified by fractional distillation at reduced pressure. Sodium
hypochlorite, 5%, was obtained from the J. T. Baker Chemical Co.
Preparative Chlorination of a-Terpineol (1_) --
For the preparative formation of the product mixture, a high concen-
tration chlorination of a-terpineol (1_) was carried out by passing chlorine
gas through a stirred mixture of a-terpineol (6 g) in 3 1. of water until
the yellow color of chlorine persisted. The excess chlorine was allowed
to escape by stirring overnight. The product mixture was removed by a con-
tinuous ether extraction, and, after drying over magnesium sulfate, the
removal of the solvent left 6 g of an oily residue. The product distribu-
tion, as determined by analytical LC, was similar to the controlled reactions
24
-------
used in the product distributions "given in Table 4. Separation was accom-
plished by column chromatography on silica gel with an initial benzene
elution and subsequent ether/benzene elutions, increasing the ether to
benzene ratio until pure ether was the eluent. The separation was monitored
by analytical LC.
B-2, a-6-Dichloro-cis-p-menthane-l, 8 diol (2) --
The column chromatography fraction corresponding to 2^ was purified by
recrystallization from chloroform, mp 138-139°; nmr (acetone-ds), 6 1.18
(s, 6, CH3), 6 1.80 (s, 3, CHa), 5 2.95 (s, 1, OH), 6 4.10 (s, 1, OH),
6 4.10 (m, 1, H2, W| * 9 Hz). The mass spectral and ir characteristics
are consistent with the dichlorodiol 2.
Analysis — Calcd for C]oHi802Cl2: c» 49.80; H, 7.52.
Found: C, 49.76; H, 7.56.
1 ,2-Dichloro-p_-menthane-8-ols (3) --
The mixture of di chlorides"! 3 corresponding to 3_ was obtained independent-
ly by dissolving a-terpineol (0.31 g, 0.001 mol) in 14 ml carbon tetra-
chloride containg chlorine (0.071 g, 0.001 mol). The solvent was removed
after 0.5 hr, and the crude product was subjected to column chromatography
conditions corresponding to those applied to the products obtained from the
aqueous chlorination. The resulting dichlorides had the expected mass
spectral, ir, and nmr characteristics, and they corresponded to those for
the product isolated from the aqueous preparative reaction.
a-2-Chloro-cjsrp-menthane-l, 8-diol (4_) —
As can be seen from Table 4, a basic heterogeneous chlorination of
a-terpineol (]_) provides the best conditions for obtaining the diequatorial
chlorohydrin 4_. In the preparative formation of 4_, 10 g (0.065 mol) of a-
terpineol was mixed with 0.5 1. of water. The mixture was stirred vigorously,
and 150 ml of a 5% sodium hypochlorite solution was added. The pH was brought
to about 8 by the dropwise addition of concentrated hydrochloric acid.
Stirring was continued for 2 hrs, and then a solution of sodium sulfite was
added to destroy excess chlorine. The aqueous mixture was extracted with
ether in a continuous extraction apparatus. The combined ether extracts
were dried over anhydrous magnesium sulfate, and the ether was removed under
reduced pressure which yielded 10 g of a yellow oil. Crystals formed upon
standing, and they were recrystallized from a 3:1 solution of chloroform:
hexane; mp 118. 5-119. 5°, nmr (DCC13), 6 1.20 (s, 6, CHj), S 1.30 (s, 3, CH3),
6 4.00 (dd, 1, fy* J2 3a = ^^ Hz and J2 3e = 4.-5 Hz)- Tne mass and 1r
spectra were in agreemeTTt with the monocnloro diol structure.
Analysis -- Calcd for C10Hi902Cl: C, 58.11; H, 9.26; Cl , 17.15.
Found: C, 58.13; H, 9.25; Cl , 17.31
B-2-Chloro-trans-p-menthane-l ,8-diol (5_) —
The peak corresponding to 5_ in the preparative acidic aqueous chlorina-
tion of a-terpineol (1) was purified by recrystallization from chloroform,
25
-------
mp 93-940, nmr (acetone-d6), 6 1.12 (s, 6, CH3), 6 1.28 (s, 3, CH3), 6 2.96
(m, 1, OH); 6 2.95 (s, 1, OH); 6 4.09 (m, 1, fi^); mass and ir spectra con-
firmatory.
Analysis — Calcd for C-|QHig02Cl: C, 58.10; H, 9.26; Cl , 17.15.
Found: C, 57.95; H, 9.08; Cl , 16.86.
6-Chloro-cj_s_-p-menthane-l,2,8-triol (6) —
This product can be isolated from either the acidic or basic aqueous
chlorination. Purification was accomplished by recrystallization from ace-
tone, mp 146-1470; nmr (acetone-de); 6 1.16 (s, 6, CHs), 6 1.77 (s, 3, CH3),
6 3.10 (m, 2, OH), «5 3.92 (m, 2, H2 & H6), 6 4.55 (d, 1, OH, J = 8 Hz);
mass and ir spectra confirmatory. This triol can also be prepared by the
hydrolysis of the dichloro diol 2. Thus when 2_ was dissolved in 1 M hydro-
chloric acid and stirred at 25QC for 48 hrs, and worked up by ether extrac-
tion, the crystalline product obtained was identical in physical and spec-
tral properties as well as mixed melting point with the triol 6_.
Analysis — Calcd for C^gOaC!: C, 53.95; H, 8.60; Cl , 15.92.
Found: C, 53.93; H, 8.75; Cl , 15.81.
m-Chloroperoxybenzoic acid epoxidation of a-terpineol (1_) --
The a-epoxide 7_ and bicyclic ether £ are best synthesized by direct
epoxidation of a-terpineol (]_). In a one-liter flask, fitted with a drop-
ping funnel and a CaClo-filled drying tube, were placed 250 ml of methyl ene
chloride and 8 g of 85% (0.04 mol) m-chloroperbenzoic acid (Aldrich Chemical
Co.). This solution was cooled in an ice-water bath and magentically stirred
while 5.5 g (0.036 mol) of a-terpineol (]_) in 100 ml of methylene chloride
was added over a two-hour period. The solution was then allowed to warm
to room temperature, and 100 ml of a water solution of 2.8 g (0.056 eq) of
sodium carbonate and 7.0 g (0.056 mol) of sodium sulfite was added dropwise
over 10 min. The water solution, after it was stirred for 15 min, was weakly
basic and gave a negative test with starch iodide paper J^ The organic
layer was separated, and the aqueous solution was extracted with two 100-ml
volumes of ether. The combined organic extracts were dried over magnesium
sulfate and filtered, and the solvent was removed at reduced pressure. A
colorless oil, 5.6 g, was recovered. The two major components in this
mixture were separated by vapor phase chromatography (10' x 3/8", 20% DEGS,
1600). The product with the shorter retention time (15 min) was a crystal-
line material that had the same physical and spectral properties and micro-
analysis as the recently reported 2-endo-cineolylol (9). Oxidation to the
corresponding ketone via a chromium trioxide pyridine complex in methylene
chloride! 5 and formation of the corresponding oxime gave a mp 138°, reported
1390.16 The product with the longer retention time (55 min) was an oil
corresponding to £-menthane-a-l ,2-epoxy-8-ol (7), bp 600 (0.2 mm); nmr
(CC14), 6 1.08 (s, 6, CHs), 6 1.25 (s, 3, CH3), 6 2.96 (d, 1, H2, 02, 3e =
4.5 Hz), mass and ir spectra confirmatory. ' ~
26
-------
Analysis — Calcd for CioHig02: c» 70-55'« H» 10-60
Found: C, 70.63; H, 10.43.
p-Menthane-e-1,2-epoxy-8-ol (8_) —
An analytical sample of the B-epoxide 8_ is most readily prepared by
the treatment of chlorohydrin 4^with base. In a 30-nrl flask was placed
0.50 g (0.0025 mol) of chlorohydrin 4_and 25 ml of water. This mixture was
cooled in an ice-water bath and magnetically stirred while 0.84 g (0.015 mol)
of potassium hydroxide was added in small batches over a six-hour period.
The mixture was then allowed to warm to room temperature, and the stirring
was continued for another three hours. The aqueous mixture was extracted
with four 25-ml volumes of ether. The ether extracts were dried over magne-
sium sulfate and filtered, and the ether was removed under reduced pressure.
A short-path distillation produced 0.35 g of an oil, bp 55-60° (0.2 mm),
nmr (DCC13), 6 1.15 (s, 6, CH3), <5 1.13 (s, 3, CH3), 6 3.06 (s, 1, H2).
mass and ir spectra confirmatory.
Analysis — Calcd for C-|oHl802: C, 70.55; H, 10.66.
Found: c, 70.48; H, 10.57.
REFERENCES
1. Simonsen, 0. L., "The Terpenes -Vol. 1, Part II," Cambridge University
Press, Cambridge, England , 1953 .
2. White, G. C., "Handbook of Chlorination," Wan Nostrand-Reinhold Company,
New York, N. Y., 1972.
3. Morris, J. C., J. Amer. Water Works Assoc.. 6J_, 769 (1971).
4. Cooke, A. H., Chem and Ind., 164 (1971)
5. Keith, L. H., Environ. Sci. Techno!., lp_, 555 (1976).
6. Maahs, H. G., L. N. Johanson and J. L. McCarthy, Tappi. 5£, 270 (1967).
7. Hrutfiord, B. F and J. L. McCarthy, Tappi, 50_, 82 (1967).
8. Matteson, M. J., L. N. Johanson and J. L. McCarthy, Tappi. |p_, 86 (1967).
9. Banks, R. C., "Isolation of Certain Toxic Components of Kraft Mill
Wastes and Attempts to Determine their Structure," Ph.D. Thesis,
Oregon State University, Corvallis, Oregon, 1969.
10. Slawinski, K., Chemik Pol ski. JJ_, 97 (1917).
11. Slawinski, K. and G. Wagner, Chem. Ber., ||, 2064 (1899).
12. Kopperman, H. L., R. C. Hallcher, Sr. A. Riehl, R. M. Carlson and R.
Caple, Tetrahedron, 3^, 1621 (1976).
27
-------
13. Royals, E. E. and J. C. Leffingwell, J. Org. Chem., 3_L, 1937 (1966).
14. Fieser, L. F and M. Fieser, "Reagents for Organic Synthesis - Vol. T,"
John Wiley and Sons, Inc., New York, N. Y., 1967, p. 136.
15. Ratcliffe, R. and R. Rodehorst, J. Org. Chem.. 35., 4000 (1970).
16. Beilstein, Band XVII, Springer Verlag (1933), p. 266.
AQUEOUS OZONATION OF a-TERPINEOL
Results and Discussion
Ozonation appears to be a viable alternative to chlorination as a means
of disinfection (Section 1 - Introduction). The effectiveness of ozone in
water purification processes is in some respects, e.g., color and taste removal,
disinfection, and reduction of BOD^»2 superior to chlorine. However, in
addition to the economic question, "Changing the disinfectant without an
intense research input to study other public health ramifications could be
a catastrophic step."3
Oust as the olefinic center in a-terpineol is very susceptible to
electrophilic attack upon aqueous chlorination, the olefinic center also
reacts readily with electrophilic ozone. This interaction usually leads
to the formation of products resulting from the cleavage of the carbon-
carbon double bond.
°3
o —.
Acids, etc. depending on nature
substituent group
Possible non-cleavage products include epoxides, glycols, primary ozonides,
normal ozonides, and tetra oxolanes.
It seems reasonable to compare the aqueous chlorination chemistry of
a-terpineol with the aqueous ozonation chemistry. The major product upon
the interaction of ozone in water with a-terpineol at either an acidic pH
of 3 or basic pH of 10 is a keto-lactone.
28
-------
H20
a-terpineol
-7—0
Keto-lactone derived from
initial alkene cleavage
The formation of the keto-lactone is consistent with the Criegee Mecha-
nism (Section 1) via intermediacy of the hydroxy hydroperoxide.
f
a-terpineol_
H20
OH
-HoO
-HoO
Lactonization
Hydroxy-
hydroperoxide
Observed
keto-lactone
An alternate mechanism for the formation of the keto-lactone via the
intermediate keto aldehyde, an expected cleavage product, cannot be ruled
out. The ketoaldehyde was synthesized via previously reported reductive
conditions in dimethyl sulfoxide^ and could be shown to be readily converted
to the keto-lactone under aqueous ozonation conditions.
Other minor oxygenated cleavage products can also be detected in the
aqueous ozonation of a-terpineol, but these products have yet to be unambi-
guously identified. It would be desirable to learn these structures to
enhance our understanding of aqueous ozone chemistry and increase our pre-
dicitive capabilities with other olefinic systems.5
29
-------
Experimental
a-Terpineol was purchased from Matheson, Coleman and Bell Manufacturing
Company and was fractionally distilled prior to use.
Procedure for the Ozonation of a-Terpineol --
Ten grams of a-terpineol (via magnetic stirring) with three liters of
distilled water in a five-liter Erlenmeyer flask. An oxygen-ozone mixture
was bubbled through the aqueous mixture with a sintered glass bubbler at
a flow rate of 30 ml/min (corresponding to about 1.5 x 10~4 moles/min of
ozone). The bubbling was continued for 7i hours at 250C. At the end of
this time a Kl-starch paper test indicated the presence of peroxidic material.
The reaction mixture was allowed to stand overnight and then was slowly
added to a 25-X 5-cm Amerlite XAD-2 column. The column was eluted with 50:50
ether/methanol, and 7.2 g of a crude product was obtained. The nmr and GC
(OV-1, 5%, 1200) indicated the major product (75%) to be the keto-lactone
a.ct-dimethyl butyrolactone. The keto-lactone was purified by recrystallising
from hexane/ether, mp 60° (lit. 610)6; ir> 1755, mo, 1375, and 1390 cnH;
ms, M+ 184, 151, 123, 111, 98; nmr 6 1.28 6(3); 6 1.45 s (3), 6 2.2-2.6 m(3).
Analysis — Calcd: C, 65.44; M, 8.86
Found: C, 65.19, M, 8.75.
REFERENCES
1. Tenney, Robert I., "Ozone, the Add-nothing Sterilant," Technical
Quarterly. ]£, 1 0973 ).
2. Blogoslawski, W. A. and R. G. Rice, ed., "Aquatic Applications of Ozone,"
International Ozone Institute Workshop, Syracuse, N.Y., 1975.
3. Stevens, Alan A. in'Proceedings of the Conference on the Environmental
Impact of Water Chlorination," Oak Ridge National Laboratory, Oak
Ridge, Tennessee, Oct. 22-24, 1975, p. 88.
4. Bozzato, G., J. P. Bachmann, and M. Pesaro, Chem. Commun., 1005 (1969).
5. Hilleren, Norman 0., M.S. Thesis, University of Minnesota, Duluth, 1977.
6. Henry, T. A. and M. Paget, J. Chem. Soc.. 70 (1928).
OLEIC ACID - AQUEOUS CHLORINATION
Results and Discussion
The reaction of oleic acid with chlorine1 in distilled water produces
a mixture of 9-chloro-10-hydroxystearic acid (1) and 10-chloro-9-hydroxy-
stearic aciJ (2) over a wide range of pH values and chlorine concentrations
(Table 5). 2
30
-------
The presence of ammonia during the chlorination not only results in
the formation of the chlorohydrin mixture but the generation of 9,10-epoxy-
stearic acid3, 9,10-dihydroxystearic acid4, and an unidentified material
that has comparable hydrophilicity to that of the glycol.
CH3-(CH2)7-CH=CH-(CH2)7-C02H (oleic acid, cis)
H
CH3-(CH2)7-C-C-(CH2)7-C02H
OH Cl
1
H H
I I
CH3-(CH2)7-C—C-(CH2)7-C02H
Cl OH
2
C12/H20
A
I CH3-(CH2)7-C—C-(CH2)7-C02H
i H H
I i r
\^ CH3-(CH2)7-C-C-(CH2)7-C02H
OH.H
The chlorination of oleic acid produces a mixture of the 9,10-10,9-
chlorohydrins under a variety of conditions. However, the change in product
distribution due to the presence of ammonia is difficult to explain, as highly
basic conditions do not produce the same results. In addition, dichloro-
stearic acid is absent even at low pH values.
A mixture of the two chlorohydrins is significantly toxic to Daphnia
magna. The LC5Q (48 hours) is 2-4 ppm, but the toxicity cannot be currently
assigned to one or both of the isomers.
Experimental
All analyses were performed on a Waters Associates ALC 201 with 2x2'
Bondpak C-18 columns of Porasil B and a differential refractive index detec-
tor. The oleic acid was obtained from Nu-check-Prep Inc., Elsian, MN, in
99.9% purity.
Procedure --
Distilled water (or water containing NH4OH) was saturated with oleic
31
-------
TABLE 6. CHLORINATION PRODUCTS FROM OLEIC ACID
NaOCI
(ppm)
5.1
2.5
10.2
10.2
5.1
2.5
1.0
.51
5.1
2.5
1.0
10.2
Initial
Oleic Acid
(ppb)
30
30
25
70
80
30
11
10
5
12
10
15
Mixed
Chlorohydrin
(ppb)
10
1
7
76
75
10
1
NR
6
10
6
18
Reaction
(*)
30±2
5±5
25±2
90±2
80±2
30±2
10±10
0
100
70±2
50±2
100
PH
11.2
11.2
11.2
6.1
6.1
6.1
6.1
6.1
1.8
1.8 '
1.8
1.8
acid and adjusted to the required pH. Sodium hypochlorite was added and
allowed to react for 15 min. After the addition of excess sodium sulfite
to stop the reaction, the reaction was acidified and extracted with dichloro-
methane ( 3 x 60 ml). The resulting solution was dried over sodium sulfate,
filtered, and evaporated in vacuo.
Threo-9,10-Dichlorostearic Acid —
The procedure of Pihlaga and Ketola2 was followed: oleic acid (1.12
g) was added to 15 ml of CC14 at room temperature. To this mixture was
added 40 ml of Cl2 saturated in CCU over a period of 15 min. After an
additional 5 minutes the Cl2 and CCl4 were removed on a rotary evaporator.
Petroleum ether (25 ml, 30-60°C) was added and the solution set aside to
crystallize at -20°C. The crystals were collected by cold filtration
[yield 0.56 g (40%) m; 36-37°C, Iiterature2 mp 37.4-377817
C_is_-9,10-Epoxystearic Acid —
To a solution of oleic acid (l.lg) in dichloromethane (28 ml) was
added a solution of 1.10 g of m-chloroperbenzoic acid in 12 ml of dichloro-
methane. The reaction mixture was stirred at room temperature for 2.5 hours
and was then washed once with approximately 100 ml of a 10% sodium sulfite
solution. The aqueous layer was discarded, the organic layer was dried over
anhydrous sodium sulfate and filtered, and the dichloromethane was removed
32
-------
on a rotary evaporator. The solid residue was crystallized from 25 ml of
acetone [yield .85-.87 g (69-70%) mp 56-57.5<>C, literature mp 59-59.5°C.3]
Threo-10-Chloro-9-Hydroxystearic Acid and Threo-10-Hydroxy-9-Chlorostearic
Acid Mixture --
The procedure was essentially that of McGhee e_^al_.4, in which concen-
trated hydrochloric acid (0.9 ml) was added to 9,10-epoxystearic acid (250
mg) in 10 ml of ether. After stirring for 45 minutes, the aqueous layer
was decanted and the ether layer was washed with water to remove the mineral
acid. The ether was dried over anhydrous sodium sulfate, filtered, and
evaporated in vacuo. After crystallization of the residue from 17 ml of
petroleum ether (30-60°C) at -20°C, the crystals were collected [yield 191
mg (67%) mp 34-37°C, literature 33-41oc]4.
REFERENCES
1. Markley, K. S., "Fatty Acids - Vol II", Interscience Pub. Co., New York,
N.Y., 2nd Ed., 1961, p. 1084.
2. Pihlaqa, K. and M. Ketota, Suomen Kemistilehti, ^3_, 21 (1970.
3. Swern, D., J. Amer. Chem. Soc.. 7Qt, 1235 (1948).
4. McGhee, J. F., W. A. Ross, B. L. Rad, W. A. Cramp and C. B. Thornton,
J. Chem. Soc.. 3108 (1962).
AQUEOUS OZONATION OF FATTY ACIDS (OLEIC AND LINOLEIC ACIDS)
Results and Discussion
The interaction of ozone with oleic acid was investigated to compare
the aqueous ozone chemistry with the aqueous chlorine chemistry. As with
a-terpineol, the reactive center in both of these reactions is the unsatura-
ted olefinic linkage.
The major products in a typical aqueous ozonation of oleic acid are
cleavage products of the olefinic linkage (Table 7). These products were
followed by a GC analysis after methylation with diazomethane. The initial
pH was about 3.9 and decreased slightly as the ozonation proceeded. The major
cleavage products are aldehydic, but the ratio of products changes because
of the subsequent autoxidation of the aldehydes with time. These cleavage
products are those anticipated from the Criegee ozonolysis mechanism (Section
1).
A non-cleavage ozonation product was also observed in the aqueous
ozonation of oleic acid. Significantly, this product was identified as
the epoxide of oleic acid (i.e., oleic acid oxide). The formation of epoxides
from olefins is always of interest because of the potential carcinogenic
properties of this functionality.1"7 Preliminary studies on the origin
of oleic acid oxide in this reaction indicate that it is not formed by
direct epoxidation with ozone of oleic acid. It appears more likely that
epoxide formation results from a subsequent peracid epoxidation of oleic
33
-------
TABLE 7. AQUEOUS OZONATION PRODUCTS OF OLEIC ACID
03 CH2N2
CH3(CH2)7CH=CH(-CH2)7C02H^ metny1at1on>
Oleic Acid (cis)
CH3-(CH2)7-CH=CH(CH2)7C02CH3 methyl oleate
CH3{CH2}7CHO n_-nonyl aldehyde
CH3{CH2)6C02CH3 methyl caprylate
dimethyl suberate
OH OH
CH3{CH2)7-CH-CH4CH2)7-C02CH3 methyl 9,10-dihydroxysterate
0
CH3{CH2}7C-CH3 n-nonyl methyl ketone
Tyia methyl insertion in n-nonyl-
aldehyde)
OHC(CH2)7C02CH3 methyl azelaldehydate
Q
CH3C(CH2)7C02CH3 methyl ketone of above via methyl
insertion
CH302C{CH2}7C02CH3 dimethyl azelate
34
-------
acid with the peracids arising from autoxidation of the aldehydic products.
The generality of this mechanistic possibility should be examined further,
0
CH3-(CH2)7-CH-CH-(CH2)7-C02H
Oleic acid oxide
0,
0
II
R-C-H
Initial aldehydic
cleavage products
R-co3H plus :r=cC
peracid olefin
R-C02H
acid
0
/\
/I
olefin oxide
The products identified in a similar aqueous ozonation study of lino-
leic acid are listed in Table 8. A number of minor products have yet to
be identified in this reaction, a reaction that is more complex than oleic
acid owing to the presence of two carbon-carbon double bands. The cleavage
products identified can be rationalized in terms of the Criegee ozonolysis
mechanism. It will be desirable, however, to identify as many of the minor
components as possible and again look for epoxide formation.
Experimental
Procedure —
The oleic acid was obtained from Nu-Check-Prep, Inc., Elsian, MN in
99.9% purity.
Oleic acid (3.70 g) was added to a two-liter Erlenmeyer flask con-
taining 1,950 ml of distilled water. The pH of the saturated oleic acid
solution was 3.9. The solution was stirred at room temperature, and an
02-0^ mixture was bubbled through the solution with a sintered glass bubbler.
The 02-03 flow rate was 30 ml/min, which corresponded to an 03 flow rate
of 30 ml/min. After 20 minutes, a 200-ml sample of the solution was removed
and put into a 250-ml Erlenmeyer flask and stirred overnight to allow the
natural decomposition to products. Distilled water (200 ml) was added to
the reaction flask to replace the volume lost. The ozonation was discon-
tinued for approximately one minute while the first sample was being removed.
After 40 minutes a second sample (220 ml) was taken; the same procedure
was used as for the removal of the first sample. After 2 hrs 10 min, a
third sample (380 ml) was taken, and after 3 hrs 35 min from the start of
the ozonation, the ozonizer was turned off. After stirring overnight at
35
-------
TABLE 8. AQUEOUS OZONATION PRODUCTS OF LINOLEIC ACID (LINOLIC ACID)
0. CH2N2
CH3(CH2)4CH=CH-CH2-CH=CH(CH2)7C02H -^ methy1ation>
CH3(CH2)4-CHO n-hexanal
0
CH3(CH2)4-C-CH3 2-heptanone
(via methyl insertion in n-
hexanal)
CH3(-CH2-)4-C02CH3 methyl hexanoate
OHC(-CH2)7C02CH3 methyl 9-oxopelargonate
CH3C(-CH2)7C02CH3 (yja_ methyl insertion in above)
CH3OC(CH2)7C02CH3 dimethyl azelate
CH3(-CH2-)4-CH=CH-CH2CHO 3-nonenal
room temperature to allow for decomposition of peroxide intermediates, the
samples were further acidified (1 ml of concentrated HC1 for samples 1-3
and 3 ml of concentrated HC1 for sample 4) and extracted with methylene
chloride (samples 1-3 with three 40-ml portions and sample 4 with four
200-ml portions of CH2C12). The four methylene chloride solutions were
then dried (anhydrous magnesium sulfate), filtered, and evaporated in vacuo.
The product recovery was as follows: in sample 1, 0.39 g; in sample 2,
0.20 g; in sample 3, 0.43 g; in sample 4, 2.23 g, resulting in a total
product recovery of 3.25 g.
In preparation for GC analysis the samples were exhaustively methylated
with diazomethane. The ether-diazomethane solution was added to the samples
until the green-yellow color of diazomethane persisted.
36
-------
The GC analysis was accomplished by spiking the samples with a set of
probable standard compounds. This was done on three different columns of
different polarity. The columns and GC conditions were: 3% carbowax 20 M
programmed from 80° to 230°C at 90/min, a 3% phenyl silicone OV-25 programmed
from 80° to 2300 at 90/min, and a silinated 5% methyl silicone OV-1 column
programmed from 1000 to 340° at 90/min.
Of the compounds used as standards, methyl oleate was purchased from
Nu-Check-Prep, Inc., octanoic acid, ivnonyl aldehyde, r^-nonylic aldehyde,
azelaic acid, suberic acid, and 9,10-dihydroxysteric acid were all purchased
from INC Pharmaceuticals, Inc., Plainview, NY. The acids were all methylated
with diazomethane before use as standards on the GC. The other standard
compounds, methyl azelaldehydate, and the methyl ketones of rnnonyl aldehyde
and methyl azelaldehydate were synthesized by the procedure indicated below.
Methyl azelaldehydate -- This compound was made by the same procedure
as used by G. Bozzato, J. Backmann and M. Pesari in making the hemiacetal
from a-terpineol.3 Methyl oleate, 24.55 g (8.69x10-2 moles) was put in
a three-necked roundbottom flask containing 950 ml of absolute methanol.
The solution was cooled to -7QOC. An 02-03 mixture was introduced into
the reaction flask through a sintered glass bubbler at a flow rate of 30 ml/
min, which corresponded to an 03 flow rate of 4.2x10-4 moles/min. The ozo-
nation was continued until the first trace of blue color (excess 03) ap-
peared. The solution was flushed with N2 for 15 minutes, then 25 ml of
dimethyl sulfide was added. The solution temperature was allowed to rise
slowly to room temperature while flushing with N2- After twelve hours of
flushing with N;? the methanol was removed in vacuo yielding 32.6 g of product.
After washing with sodium bicarbonate, samples of n-nonyl aldehyde and methyl
azelaldehydate were obtained in >95% purity (by GC~J by vacuum distillation.
Nonanal was distilled over at 3QOC (l.Q mm) and methyl azelaldehydate at
78-820C (0.9 mm). The structures were confirmed by nmr and ir.
Methyl ketones of nonanal and methyl azelaldehydate -- Diazomethane
was added to each of two 250-ml Erlenmeyer flasks. One flask contained 1 g
of nonanal and the other contained 1 g of methyl azelaldehydate. The solu-
tions were stoppered loosely and allowed to stand overnight (15 hours),
after which the products were concentrated in vacuo. Nmr analysis showed
the product to be the methyl ketones of the corresponding aldehydes. The
purity of the nonylmethyl ketone was >90% and that of the methyl ketone
of methyl azelaldehydate was >85% (by GC).
Methyl oleate oxide — Oleic acid (1.16 g) in 28 ml of dichloromethane
was added to a solution of m-chloperbenzoic acid (1.10 g) in 12 ml of
dichloromethane. The mixture was stirred at room temperature for 2.5 hrs
and then washed once with 100 ml of 10% sodium sulfite solution. The aqueous
layer was discarded and the organic layer dried. The solvent was removed
in vacuo leaving a solid residue that was crystallized from 25 ml of acetone,
mp 56-57.5°C (lit. mp 59-59.5°C). The ci^-9,10-epoxystearic acid was then
methylated with diazomethane to methyl oleate epoxide.
The linoleic acid was obtained from Nu-Check-Prep, Inc., Elsian, MN in
99.9% purity.
37
-------
Procedure --
Linoleic acid (1.20 g) was added to a two-liter Erlenmeyer flask con-
taining 1,950 ml of distilled water. The initial pH of the saturated lino-
leic acid solution was 4.3. The ozonation was carried out at room temperature
and with a 02-03 flow rate of 30 ml/min corresponding to an ozone flow rate
of 7.8xlO~5 mole 03/min. The pH at the end of the ozonation was 3.8. The
sampling and extraction procedure was the same as that described for oleic
acid, the only difference being that the peroxide intermediates were given
three days to decompose instead of one. A test with KI starch paper in-
dicated that decomposition was not complete after one day. The sampling
and extraction procedures used on the three samples taken were as follows:
Amount H20
Sample Time ozonated sol removed
1
2
3
Amount
20 min
50 min
165 min
350
350
1,950
Extracted Amount product
with removed
200 4 fractions 0.25 g
200 4 fractions 0.25 g
800 4 fractions 0.68 g
Total product recovery 1.18 g
The samples were then methylated with diazomethane in preparation for GC
analysis.
REFERENCES
1. Van Duuren, B. L., J. Nat. Cancer Inst., 3g, 1213 (1967).
2. Van Duuren, B. L., J. Nat. Cancer Inst., 37^, 825 (1966).
3. Van Duuren, B. L., J. Nat. Cancer Inst., 35^ 707 (1965).
4. Van Duuren, B. L., J. Nat. Cancer Inst.. 3J_, 41 (1963).
5. Boyland, E. and W. H. Down, Europ. J. Cancer, 7_, 495 (1971).
6. Van Duuren, B. L. and B. M. Goldschmidt, J. Med. Chem., 9_, 77 (1966).
7. Weil, C. W., J. Industrial Hygiene. ££, 305 (1963).
CHLORINATION OF RESIN ACIDS
Results and Discussion
Diterpene "resin acids" (Ci^gCOgH) represent an additional compound
type often present in waters subjected to aqueous chlorination (e.g., the
bleaching of wood products or the disinfection of certain natural or in-
dustrial waters).
38
-------
The various chlorination products referred to in the following discus-
sion of the chlorination of dehydroabietic and abietic acids are graphically
presented in Figure 2. Numbered compounds in the text can be identified
by reference to the figure.
Chlorination of dehydroabietic acid (Compound 1) with a large excess of
chlorine gave products that could not be separated by crystallization or
silica gel chromatorgraphy. The reaction mixture was, therefore, converted
to the methyl esters with diazomethane, which in turn led to the isolation
of the esterified Compounds 3 and 4 by fractional crystallization. Low
concentration chlorination (6 equivalents) of dehydroabietic acid gave
products identical to those from high concentration chlorination as shown
by high pressure liquid chromatography (HPLC). The chlorine uptake (i.e.,
yield of products) was quite high (38% product formation).
Chlorination of abietic acid (pH 2) (Compound 2) and subsequent column
chromatography gave rise to one major and two minor product-containing
fractions. The major fraction was esterified with diazomethane and then
separated into two components by preparative layer chromatography (PLC).
One of these was assigned structure 5, and the other was shown to be iden-
tical to Compound 4.
Compound 5 exhibited a characteristic AB pattern for the olefinic pro-
tons, but these were absent in Compound 4. On the other hand, Compound 4
possessed a one-proton quartet expected for the rigid ring proton of the
type Ar-CJi-Cl-CH2-. The two aromatic protons of Compound 3 occurred as a
two-proton singlet in CDCl?, and in acetone two one-proton singlets were
observed. Thus, the chlorine must be placed on the ring so that an AB
pattern cannot be observed in the nmr spectrum.
Structures 3, 4 and 5 were also established by interconversion. Com-
pound 5, when treated with HC1 gas, gave Compound 4. Treatment of Compound
4 with Cl2 gas gave Compound 3.
One of the polar compounds formed in the high concentration and low pH
chlorination was not observed (HPLC) when the reaction was carried out in
dilute solution with one equivalent of chlorine. The empirical formula
suggests a mono epoxy chlorohydrin of the type 7. When the chlorination
was carried out as described in the literature for dichlorodihydroxyabietic
acid (Compound 6) the major product was identical to the one formed in the
chlorination carried out under mild conditions at pH 2.
The acids 8 and 10 corresponding to esters 5 and 3 respectively were
also prepared by independent synthesis. Mercuric acetate oxidation of
abietic acid followed by basic hydrolysis gave the olefin 8. Ferric-chloride
-catalyzed chlorination of Compound 9 gave dichlorodehydroabietic acid
(Compound 10) in high yield. The chlorination product, Compound 9, of
dehydroabietic acid in the absence of Feds was the monochlorinated isomer,
which could be purified on a silica column.
39
-------
, C02H
1
Dehydroabietic
acid
D C12
2) CH2N2
C02CH3
2) CH2N2
C02H /
OH
Cl
Figure 2. The chlorination of dehydroabietic acid and abietic acid.
40
-------
Cl2/FeCl3
-C12
C02H
Cl
C02H
pH 10
Figure 2.
11
9 18
(minor) (minor)
2
The chlorination of dehydroabietic acid and abietic acid
continued.
41
-------
When the chlorination of abietic acid was carried out at pH 10, Compounds
8 and 9 were formed as minor products, with the major products being the
diepoxide (Compound 11) and an aromatized compound tentatively assigned
structure 12. Table 9 outlines the overall product distribution with pH.
TABLE 9. PERCENTAGE OF COMPOUNDS FORMED BY CHLORINATION OF ABIETIC ACID
AT VARIOUS pH VALDESa.b
Compound
Diepoxide (11)
Olefinic (5)
Monepoxide (12)
Monochloride (9)
Polar component (7)
2
0
66
0
12
22
4
0
72
0
9
19
PH
6
4
71
13
6
6
8
30
4
52
12
0
10
32
4
50
19
0
Retention
8
10
13
15
20
time
.5
.1
.2
.2
.5
(min)
a Reaction conditions: 1 £. water containing 10 mg of abietic
acid treated with two equivalents Cl£ as NaOCl for 0.5 hr.
b HPLC conditions: Solvent, 4% j_-propanol, 44% hexane,
column, 3 porasil columns; flow rate, 1 ml/min.
52% ether;
Experimental
Dehydroabietic Acid Purification --
Technical dehydroabietic acid (5 g) was dissolved in acetone (10 g),
and 2-amino-2-methyl-l-propanol was added. Crystals appeared immediately,
and the mixture was allowed to stand for 24 hours. The salt was recrystal-
lized from ethanol (4.1 g, mp 190-191°). Saturated boric acid (5 ml) was
then added to a suspension of the salt in ether. After stirring for one
hour, the ether layer was separated and washed with water. Evaporation of
the ether gave dehydroabietic acid that was crystallized from ethanol water
(4/1) (3 g, mp 1720C, lit. 171-173o2); nmr (CDCls) 6 1.23 (s, 3, CHj),
6 1.17, (s, 3,
Abietic Acid Purification —
Technical abietic acid (24.5 g) was dissolved in acetone (37.5 ml),
and diamylamine (12.7 g) was added slowly to the hot solution. Upon cooling
to room temperature, crystals formed and the mixture was cooled overnight.
The solvents were removed by filtration, and after four recrystallizations
from acetone the salt melted sharply at 134°. This salt (5.4 g) was dis-
solved in 40 ml of ethanol and cooled in the refrigerator. Acetic acid (1.4
ml) and water (20 ml) were added, and the mixture was allowed to remain cold
for an additional 24 hours. The resulting abietic acid amounted to 3.5 g
42
-------
[mp 170-1720, m. 171-173°2, nmr (CDC13) 6 1.04 (s, 3, CHo), 6 .94 (s, 3,
6 .82 (s, 3,
High Concentration Chlorination of Dehydroabietic Acid --
Dehydroabietic acid (0.5 g) was dissolved in ether (10 ml) and mixed
with water (500 ml) while chlorine gas was passed through the solution for
one-half hour at a flow rate of 20 ml/min. After standing for twelve hours,
the mixture was extracted with two 100-ml portions of ether. Evaporation
of the ether left an oily residue (0.6 g). Attempts to crystallize or sepa-
rate the products by thin layer chromatography failed.
The reaction mixture was then methylated with excess diazomethane in
ether. Evaporation of the solvent left a solid glass, which was dissolved in
a minimum amount of hot ethanol . After standing in the refrigerator over-
night, the crystalline product was collected by filtration. Compound 3,
11 mg, mp 138-14QOC; nmr (CDC13) <5 7.12 (s, 2, aromatic), 6 3.67 (s, COOCfa),
6 3.38 (q, 1, ArCHCl), 6 1.29 (s, 6, 2 C^), 6 1.18 (s, 3, C^), 6 1.16 TS,
3, CHs); nmr (acetone DS) 6 7.19 (s, 1, aromatic), 6 7.34 (s, 1, aromatic).
The filtrate obtained from the isolation of Compound 3 was evaporated to dry-
ness, and the residue was dissolved in a minimum amount of hot ether. A
crystalline solid formed after the ether evaporated at room temperature.
Recrystallization gave Compound 4, 120 mg, mp 110-111°, nmr (CDCla) 6 7.00
(s, 3, aromatic), 6 3.67 (s, 3, COOCHJ,
-------
that was shown by mp and the mixed mp to be identical to Compound 3.
Interconversion of Compounds 4 and 5 --
Compound 5 (100 mg) was dissolved in methylene chloride (15 ml) and cool-
ed at 0° while anhydrous HC1 gas was bubbled through the mixture for 15 mi-
nutes. The mixture was stoppered and allowed to stand in the freezer over-
night. The partially crystallized mixture was triturated with ether, and
a crystalline product (6 mg) was collected that was shown by mp and mixed mp
to be identical to Compound 4. The reaction was observed (nmr) to be 50% com-
plete.
Low Concentration Chlorination of Dehydroabietic Acid --
A saturated solution of dehydroabietic acid (5.3 mg/1, 1.70 x 10"5m)
was treated with 7.3 mg of Clg (6 equivalents), for 0.5 hr. Sodium thiosul-
fate was added, and the reaction mixture was extracted twice with ether.
The residue was treated with diazomethane and subjected to liquid chromato-
graphy with cyclohexane/chloroform (25/1). The retention times of pure sam-
ples of the methyl esters of dehydroabietic acid, Compound 3, and Compound
4 were respectively 7.3, 8.3 and 11.6 minutes at a flow rate of 0.3 ml/min.
Known 50/50 mixtures of the compounds showed nearly identical response to
refractive index measurements. The reaction mixture contained unreacted
starting material, Compound 4, and Compound 3 in amounts of 62%, 32% and
6% respectively.
Independent Synthesis of Compound 8 --
Red mercuric oxide (1.43 g) was heated in acetic acid (3.3 ml) and ace-
tic anhydride (0.66 g) for 1.5 hours. At the end of this time, abietic
acid (1 g) in acetic acid (3.3 ml) was added, and the mixture was refluxed
for 1.5 hours. The mixture was cooled, and the metallic mercury was removed
by filtration through a sintered glass funnel. The filtrate was mixed with
water (50 ml) and extracted with two 20-ml portions of ether. The residue
obtained by evaporation of the ether was dissolved in a minimum amount of hot
methanol, allowed to cool to room temperature and then placed in the freezer
overniqht. The crystalline acetate, Compound 13 (0.32 g) was removed by fil-
tration; mp 203-2040, lit.3 202-203°C.
Compound 13 was boiled in a 1 h[ solution of KOH in ethanol (20 ml)
for two hours. At the end of this period, the mixture was cooled and made
slighly acidic with concentrated HC1. The ethanol was evaporated, and the
residue was dissolved in ether. The organic layer was washed with water,
and evaporation of the solvent left a solid glass which was dissolved in
hot ethanol. The ethanol was cooled to give a mp 170, lit.3.
Isolation of Compound 9 --
The chlorinated reaction mixture of dehydroabietic acid showed no sepa-
ration on tic. However, a column chromatography was run (90 g silica gel)
with benzene/acetone/acetic acid (400ml/10ml/40drops) at a flow rate of 20
drops/min. Analysis of the nmr spectra showed that fractions 25-42 con-
tained only the monochloride, Compound 9. A sample of the monochloride that
was with excess diazomethane had the same melting point as Compound 4.
44
-------
Independent Synthesis of Compound 10 --
Compound 9 (0.1 g) was dissolved in carbontetrachloride (15 ml), and
a catalytic amount of ferric chloride was added. Chlorine was bubbled
through the solution for one minute and the mixture was stoppered and
allowed to stand for three hours. The mixture was then extracted with
two 10-ml portions of water, and the carbon tetrachloride was evaporated
to give a product that was assigned structure 10 by its nmr spectrum.
Alternate Synthesis of the Polar Abietic Acid Chlorination Product (Rf 0.43)
— Literature Synthesis of Dichloro Dihydroxy Abietic Acid —
Abietic acid (0.3 g) was dissolved in CHC13 (5 ml) and acetic anhy-
dride (0.33 ml). A solution of sodium acetate (2.5 g) in sodium hypochlo-
rite (5%, 5.1 ml in 8 ml of water) was added, and the mixture was allowed
to stir for one hour. The organic layer was then extracted with two 10-ml
portions of water, and the CHC13 was evaporated. The residue (0.35 g) was
subjected to column chromatography (45 g silica gel 60) with benzene/acetone/
acetic acid (40/1/8 drops). The chromatography was run at a flow rate of
20 drops/min, and fractions were collected every 25 min. Fractions 35-48
contained 0.15 g of the compound (Rf 0.43) that had been isolated from the
direct chlorination of abietic acid (pH 2). The literature^ reported the
major product of this reaction to be dichlorodihydroabietic acid.
Reaction of Abietic Acid with Sodium Hypochlorite at pH 10 —
Abietic acid (0.5 g) was dissolved in three liters of water containing
sodium hypochlorite (9.8 ml of 5%, 4 mole, 2 eq.), and the pH was adjusted
to 10 with sodium hydroxide. After the mixture stirred for one hour, the pH
was lowered to 5.8 with dilute HC1, and the mixture was extracted with two
250-ml portions of ether. The ether was evaporated, and an oil (0.5 g)
was obtained. The residue obtained from four such reactions (2.3 g) was
subjected to column chromatography on silica gel 60 column (90 g). The
flow rate was 18 drops/min, and seventy fractions were combined and con-
tained respectively 0.23 g, 0.41 g, and 0.1 g. Fraction C was shown by nmr
to contain olefinic Compound 8 and a small amount of monochlorodehydro-
abietic acid Compound 9. Fraction A (Compound 11) was dissolved in a
minimum of hot ethanol, and the crystalline material was removed by fil-
tration: mp 150-153°; nmr (CDC13) 6 1.20 (s, 3, CH3), 6 1.01 (s, 3, CH3),
6 0.87 (s, 3, CH3),
-------
4. Lamprecht, M. P. R., Inst. Forestal Invest. Experiencias (Madrid),
2g, 1 (1949).
CHOLESTEROL
Results and Discussion
Chloresterol (I) was subjected to aqueous chlorination at a number of
pH values and at both high and low concentrations of chlorine. The pro-
ducts that formed corresponded to known samples of the 5-a-chloro-3B, 63-
diol (II) (the anticipated chlorohydrin^) and the a-epoxide (III). Other
possible products such as the dichlorides, the isomeric chlorohydrin, and
the triol'»2 were not observed.
-\
The chlorination products of cholesterol follow the expected stereo-
chemical patterns characteristic of the rigid steroid system whereby the
incoming reagent approaches from the less hindered (i.e., lower) a side.
The formation of the epoxide in this situation (as with terpineol) may well
be due to the presence of the six-membered ring in the presumed intermediate
chlorohydrin which would maintain the molecule in a proper conformation for
subsequent epoxide formation.
The formation of only two products in the present experiment differs
considerably from the results reported by Lindgran^. Lindgran observed
no epoxide, but other compounds (including ketonic oxidation products)
were isolated. The difference in experimental conditions appears to be
the most significant difference in the two studies. The reaction was per-
formed in these laboratories in dilute aqueous media, whereas the earlier
study was carried out at higher concentrations in aqueous butanol.
46
-------
TABLE 10. PERCENTAGE OF COMPOUNDS FORMED BY CHLORINATION OF CHOLESTEROL
AT VARIOUS pH VALUES
Summary of Results
pH Cholesterol (1) Chlorohydrin (2) a-Epoxide (2)
2.2 20% 80%
4.0 70% - 30%
7.2 80% 20%
10.0 85% 10% 5%
Experimental
The cholesterol was recrystallized from methanol before chlorination.
Reaction Procedure --
A stock solution of cholesterol in water (10 liters) was made up by
saturating the water with cholesterol and stirring for a week to assure
saturation (2.6 ppm). The cholesterol solution was filtered to remove
excess cholesterol, and the pH was adjusted by addition of HC1. The NaOCl
was added at approximately two millimole of NaOCl to one millimole of
cholesterol. The reaction mixture was stirred and allowed to run for 20
minutes. Sodium sulfite was used to stop the reaction (10 to 1 excess).
The reaction mixture was then extracted with ether, dried over magnesium
sulfate, and evaporated. The product distribution was analyzed by high-
pressure liquid chromatography.
5a-chlorocholestan-3B, 66-diol2 —
To 8.0 g cholesterol in 100 ml of ether was added 35 g Ca(OCl)* in
600 ml of H20 with stirring. Then 15 ml of acetic acid was cautiously
added, and the mixture stirred for 30 minutes. The ether was washed with
sodium sulfite solution and water and dried over magnesium sulfate. The
ether was evaporated, and the product was recrystallized with MeOH (mp
145-1470C).
a-Epoxide^ --
The a-epoxide was made by a modification of the published procedure.
A cold solution of m_-chloroperoxybenzoic acid (2.175 g) in ether (35 ml)
was added to cholesterol (3.185 g) in 20 ml of dry ether and allowed to
stand for 24 hours at -5°C. The solution was washed with ice water, sodium
bicarbonate, water 5% ferrous sulfate solution, and water. Then ether was
dried over magnesium sulfate, evaporated, and recrystallized from MeOH
(mp 141.0-142".OOC).
Dichloride4 —
Two grams of cholesterol was dissolved in 20 ml of CHC13 containing
47
-------
0.08 g antimony trichloride and cooled to -20°C. In another flask 200 ml
of CHClg were saturated with chlorine by bubbling chlorine gas into the
solution. The chlorine solution was added to the cholesterol solution
until the yellow color remained. The chloroform solution was washed suc-
cessively with sodium carbonate solution, 1 N HC1 and water and dried over
magnesium sulfate. The product was recrystaTlized from MeOH-ethyl acetate
(mp 136.5-137.50C).
6e-chlorocholesten-33, 5a-diol5 —
To one gram of cholesterol epoxide in 50 ml of tetrahydrofuran was
added 1 ml of concentrated HC1 with stirring. After 12 hours the solution
was extracted with ether, washed with water, and dried over magnesium sul-
fate (mp 138.5-140.0° from methanol). The 5a-chlorocholestan-3e, 6a-diol
isomer can be obtained from the recrystallization solvent in small quantities.
REFERENCES
1. Lindgran, B. 0., Acta Chem. Scan., 21, 1397 (1967); B. 0. Lindgran,
Svensk Papperstian. 70, 532 (1967).
2. Fierser, L. F. and S. Rajagopalan, J. Amer. Chem. Soc., 71., 3938 (1949).
3. Villotti, R., C. Djerassi and H. J. Ringold, J. Amer. Chem. Soc., 81,
4566 (1959). —
4. Barton, D. H. R. and E. Miller, J. Amer. Chem. Soc., 72_, 370 (1950).
5. Nori, S., J. Chem. Soc. Japan, 64_, 981 (1943).
PREPARATIVE METHODS FOR CHLOROPHENYL PHENOL SYNTHESIS
Results and Discussion
The synthesis of chlorophenyl phenols was initiated to provide addi-
tional examples of phenols suitable for inclusion in our study of chemical
structure-biological activity relationships. Moreover, the availability of
such a synthetic capability would have value for the preparation of possible
polychlorinated biphenyl (PCB) metabolic products!, which as examples of
chlorophenyl phenols would be expected to exhibit herbicidal, fungicidal
and antibacterial activity.2"'5
The effort expended on the synthesis of these phenols was limited
because of the insolubility of the polychlorinated products in water and
the accompanying difficulty in pursuing valid toxicity measurements.
Several simple chloro analogs of 4-phenylphenol were synthesized by
methods (or variations of methods) previously reportedl-4»lo-19.
48
-------
Cl-
CC14
Cl Cl
2.
3.
Ac20
Cl2/CCl4
KOH/EtOH
Cl-
CC1,
Cl
Figure 3. Chiorophenylphenol synthesis.
A direct translation of the above procedure to 2-phenylphenol was
unsuccessful because the anticipated chlorination in the ring bearing the
acetate did not occur. In addition, diazo coupling with a chlorophenol
only produced the diazine. An alternative approach to direct chlorination
was, therefore, used20-24} in which the phenolic-OH was introduced in the
last step through nitration, reduction, diazotization, and hydrolysis of a
biphenyl already bearing the desired chlorination pattern. This approach
took advantage of the ready availability of the required chloroanilines and
the anticipated nitration of the biphenyl nucleus in the ring not bearing
the chlorines.
Experimental
1(3',4'-dichlorophenyl)-3,3-dimethyltriazene —
To a one-liter beaker containing 32.4 g (0.2 mole) of recrystallized
3,4-dichloroaniline were added 25 ml of concentrated hydrochloric acid and
50 ml of water. This mixture was heated (85°C) to dissolve the 3,4-di-
49
-------
2. HN02
chloroaniline. At the point where solution was achieved, another 55 ml
of concentrated hydrochloric acid was added. The solution was immediately
cooled to -3 to -5°C. A thermometer was placed in the beaker, and stirring
was continued. A solution of 144 g (0.21 mole) of sodium nitrite was added
dropwise and below the surface by means of a long-stemmed separatory funnel
at a rate to maintain the temperature below 0&C. The stirring was continued
for 15 minutes after the sodium nitrite addition was complete. A urea
solution (1 g in 5 ml of water) was slowly added to decompose the excess
nitrite, and stirring and cooling were continued.
A solution was prepared containing 250 ml of water and 87 g of sodium
carbonate. To this was added enough crushed ice to lower the temperature
to 10°C, 27.0 g of dimethylamine (Aldrich; 40% aqueous) was added, and then
the diazonium salt solution was added to this solution dropwise and under
the surface. The temperature was maintained at 10°C by the addition of
more ice. After the addition was complete, stirring was continued for 15
minutes. The triazene precipitated and was recrystallized from ethanol
(95%) to yield 32 g (73%).
1(2',4'-dichlorophenyl)-3,3-dimethyltriazene and 1 (3',5'-dichlorophenyl)-
3,3-dimethyltriazene --
These two compounds were prepared in the same manner as described
above. 2,4-dichloroaniline resulted in an 89% yield, 3,5-dichloroaniline
gave an 85% yield.
A mixture of 1.09 g (5 moles) of l-(3%4'-dichlorophenyl)-3,3-dimethyl-
triazene and 3.2 g (20 moles) of 2,6-dichlorophenol was heated as liquids
(5 hours). Thin-layer chromatography indicated one major product.
3,4-dichlorobiphenyl —
To a 250-ml round bottom flask was added 11.4 g (0.052 mole) of 1-
(3',4'-dichlorophenyl)-3,3-dimethyltriazene, 16.2 g (0.07 mole) of anhy-
drous camphorsuIfonic acid, and 100 ml of benzene. This mixture was reflux-
50
-------
ed for two hours, allowed to cool, and poured into 200 ml of cold water.
The benzene layer was separated, washed with saturated aqueous sodium
bicarbonate, then washed with water, and then dried over sodium sulfate
and concentrated (9.5 g).
The concentrated material was dissolved in petroleum ether (30-60°) and
filtered through a silica-gel column (80 g) to yield 7.2 g (62%) of 3,4-
dichlorobiphenyl.
2,4-dichlorobiphenyl and 3,5-dichlorobiphenyl --
These two compounds were prepared in the same manner as described above.
The yields were 70% and 65% respectively.
2- and 4-(3',4'-dichlorophenyl)-nitrobenzene --
To a 100-ml round-bottom flask were added 12.2 g (0.051 mole) of 3,4-
dichlorobiphenyl, 30 ml of glacial acetic acid and 15 ml of concentrated
nitric acid. After refluxing for eight hours (1300-135°), the reaction
mixture crystallized upon cooling. The crystals were washed well with water
and then recrystallized from ethanol to yield 12.3 g (10%) of a mixture
of the ortho- and para-substituted compounds. Thin-layer chromatography
and nmr indicated the para- substituted product to be the major component.
2- and 4-(3',4'-dichlorophenyl) aniline —
Procedure A -- To a 25-ml round-bottom flask were added 500 rug (1.9
moles) of the ortho/para (3',4'-dichlorophenyl) nitrobenzene mixture, 5 ml
of methanol, 2 ml of concentrated hydrochloric acid and 1 g of iron powder.
This mixture was allowed to reflux for 12 hours, cooled, then extracted
with benzene, washed with saturated sodium bicarbonate and then with water,
dried over sodium sulfate and concentrated to yield 350 mg of oil. Thin-
layer chromatography indicated no starting material and prep TLC yielded
150 mg (34%) of 4-(3',4'-dichlorophenyl) aniline.
Procedure B -- To a 250-ml Parr hydrogenation bottle were added 5.2 g
(19 mmo'les) of the ortho/para-(3",4"-dichloropheny1)-nitrobenzene mixture,
50 ml of absolute ethanol and 300 mg of 10% Pd/C. The bottle was placed in
a Parr hydrogenation apparatus and was shaken until hydrogen uptake was
complete (15 min). The solution was filtered, and the ethanol was removed
to yield 4.5 g of oil. Thin-layer chromotography indicated two major spots.
2-phenylphenyl acetate --
To a 250-ml round-bottom flask set up for reflux were added 42.5 g
(0.25 mole) of 2-phenylphenol, 76 g (0.75 mole) of acetic anhydride, and
0.6 g of sodium acetate. This mixture was refluxed for three hours, cooled,
poured into 750 ml of water, and allowed to stand overnight. The aqueous
mixture was extracted three times with 100-ml portions of ether. The organic
layers were combined, washed with water, saturated sodium bicarbonate, and
again with water, dried over sodium sulfate and concentrated to give 50 g
(54%) of pale yellow oil.
2-(2',4"-dichlorophenyl)-phenol --
4 To a flask fitted with a gas bubbler were added 21.3 g (0.1 mole) of
2-phenylphenylacetate in 300 ml of carbon tetrachloride and a trace of
51
-------
iodine. To this solution 15.6 g (0.22 mole) of chlorine was slowly bubbled
over a period of 2.5 hours. Stirring was continued for two hours, and the
solvent was removed.
This crude material was placed into a 500-ml round-bottom flask, and
150 ml of ethanol, 150 ml of water, and 50 g of potassium hydroxide were
added. The mixture was refluxed for 15 minutes, allowed to cool, poured
into 100 ml of water and acidified, and the oil was separated. The oil
was extracted with ether and dried in the usual manner. A silica-gel
column was prepared (100 g), and the material was eluted in 10 carbon
tetrachloride in petroleum ether (30-60). The first fraction off the column
was crystalline (mp 144-1450), n.5 g (48% yield).
REFERENCES
1. Block, W. D. and H. H. Cornish, J. Biol. Chem.. 234. 3301 (1959).
2. Lutz, K. and H. Hemmi, Chem. Abstr., 5£, P4201b (1958).
3. Ashikaga, M., Okayama-Igakkai-Fasshi, 6£, 965 (1954); Chem. Abstr., 52_,
10391d 0958 ). — —
4. Polak, 0., Chem. Abstr.. 52_, P20864f (1958).
.5. Viel, 6., et al., Bull. Soc. Chim. Biol.. 40_, 1617 (1958); Chem. Abstr..
H, 5573 0959). —
6. Sus, 0, K. Holler and H. Heiss, Chem. Abst., 54_, P1473g (i960).
7. Fsolnai, T., Biochem. Pharmacol., 5_, 1 (1960).
8. Josephs, M. J., Chem. Abstr.. 5£, P1798e (1962 ).
9. Thizy, A., Ind. Chim. Beige, Suppl.. 2, 455 (1959); Chem. Abstr.. 54,
12261b (19607: ~ ~~
10. Stanek, J. and 0. Drahonovsky, Coll. Czech. Chem. Comm., 30, 1936
(1965); Chem. Abstr.. 63_, 3358f (1965). —
11. Monsanto Chemicals Ltd., French Patent #1,436,883, Chem. Abstr .67_ (1967).
12. Peacock, W. L., Chem. Abstr.. 69, P44740v (1968).
13. Sato, T. and M. Katsumi, Chem. Abstr., 72^ P33207m (1970).
14. Winicov, M. W. and W. Schmidt, Chem. Abstr.. 7_5, P75339z (1971 ) .
15. Dewar, N. E. and S. I. Razio, Chem. Abstr.. 74_, P24995g (1971).
16. Savoy, C. M. S. and J. L. Abernethy, J. Amer. Chem. Soc., 64, 2219
(1942). —
52
-------
17. Savoy, C. M. S., et al.. J. Amer. Chem. Soc., 65_, 1464 (1943).
18. Colbert, J. C., W. Meigs and B. Mackin, J. Amer. Chem. Soc.. 56, 202
(1934). —
19. Weissberger, A. and I. F. Salmineu, J. Amer. Chem. Soc., 67^, 58 (1945),
20. Bachmann, W. E. and R. A. Hoffmann, "Organic Reactions, Vol. II", J.
Wiley and Sons, Inc., New York, N.Y., 1944, p. 239.
21. Colbert, J. C. and R. M. Lacy, J. Amer. Chem. Soc., 68_, 270 (1946).
22. Huisgen, R. and H. Nakaten, Ann. Chem.. 586. 84 (1954).
23. Gilman, H., "Organic Synthesis" Coll- Vol. I, 0. Wiley and Sons, Inc.,
New York, N.Y., p. 396 (1933).
24.
24. Horning, E. C., "Organic Synthesis" Coll. Vol. Ill, J. Wiley and Sons,
Inc., New York, N.Y., p. 718 (1953).
53
-------
SECTION 5
BIOLOGICAL STUDIES
STRUCTURE-TOXICITY CORRELATIONS OF PHENOLIC COMPOUNDS DETERMINED WITH
DAPHNIA MAGNA
Results and Discussion
To investigate the environmental impact of the products derived from
the aqueous chlorination and ozonation of organic compounds known to exist
in waste effluents, a rapid and reliable bioassay was needed to determine
which compounds were highly toxic. Daphnia magna was chosen for the screen-
ing procedure because this animal is relatively easy to rear and to manipu-
late, and it is responsive to added toxicants^»6 such as to be considered
generally representative of aquatic invertebrates.
Phenols were studied because of their frequent appearance in effluents
and their availability as pure samples bearing systematic structural varia-
tions. In addition, a significant amount of mechanistic and toxicity data
is available on phenol itself?-'2. Phenols assume added significance when
it is recognized that all the compounds listed in Table 11 have the ability
to incorporate chlorine over a wide range of pH and concentration.^
The structure-activity correlation was investigated by using the pro-
cedures developed by Hansch.14 This methodl5-19 attempts to correlate the
biological response (BR) with two parameters, the relative partition coef-
ficient IT and the Hammett electronic substituent constant a. The parti-
tion coefficients are evaluated by using an r^-octanol-water system, and IT
is defined as log Px-log Pu, where P^ is the partition coefficient for
phenol itself, and Px is the partition coefficient for a derivative.
The general form of the Hansch equation is shown below in Eq. (1).
BR = a2 + binT0 + CTTTT02 + do + e (1)
One can expect to see simplified versions of Eq. (1) depending on the
relative importance of the parameters TT and a and the constant TTQ. These
modifications are given in Eq. (2) through (6).
BR = k-|TT + k£ (2)
BR = k-jTi2 + J^TT + k3 (3)
BR = kia + k£ (4)
BR = kp + k20 + k3 (5)
54
-------
O 15 30 45 60 75 90 105
Figure 4. Log of the percent survivors vs. time, using o-cresol
5 6 7 8 9 10
Log molar concentration (-1O )
Figure 5. Probability of survival vs. log of the molar concentration,
using o-cresol.
55
-------
TABLE 11. TOXICITY OF SUBSTITUTED PHENOLS TO DAPHNIA MAGNA
log lie
Functions
3-Methoxy
2-Methoxy
4-Methyl
2-Methyl
H
2,6-Dimethyl
4-Nitro
2-Chloro
4-ChIoro
4-Bromo
2,4-Dinitro
4-Phenyl
2,4-Dichloro
2,4,6-Tribromo
Sa
0.12
-0.27
-0.17
-0.17
0.00
-0.34
1.22
0.23
0.23
0.23
2.02
0.01
0.46
0.83
Sn
0.10
0.22
0.48
0.49
0.00
0.88
0.50.
0.69
0.93
1.13
0.06
1.91
1.62
2.91
ZF
0.26
0.26
0.04
0.04
0.00
0.08
0.67
0.41
0.41
0.44
1.34
0.08
0.82
1.32
SK
-0.51
-0.51
-0.13
-0.13
0.00
-0.26
0.16
-0.15
-0.15
-0.17
0.32
-0.08
-0.32
-0.51
Calcd. Observed A log lie
3.574
3.634
3.907
3.912
3.731
4.042
4.387
4.167
4.287
4.388
4.572
4.673
4.710
5.461
3.480
3.680
3.709
3.835
3.904
4.036
4.219
4.238
4.426
4.463
4.591
4.667
4.795
5.403
-0.094
0.046
-0.198
-0.077
0.173
-0.006
-0.168
0.071
0.139
0.075
0.019
-0.005
0.085
-0.058
c (observed)
3.31-
2.09-
1.95-
1.46-
1.02-
9.20-
6.04-
5.78 •
3.75-
3.44-
2.56-
2.15-
1.60-
3.95-
10-*
10-*
10-*
10-*
10-*
10-*
io-5
10-*
10-*
10-*
10- s
10-*
10-*
io-s
56
-------
BR = kp2 + k2Tr + kso + k4 (6)
Equation 4 considers the unlikely case where there is no dependency
on TT. A dependency on a only would suggest a situation more easily visualized
in an in vitro system rather than an in vivo oneJ6
Hansch et^al_.20 recently reported using a new set of parameters, F and
R, which attempted to separate the inductive (F) and resonance (R) components
of the substitutents. Because of the method by which F and R have been
derived (i.e., from am and on) and the demonstration of the additive nature
of o for use in structure-activity correlations, 9 it appears reasonable to
assume that F and R are additive. We utilized this argument and included
the following two additional equations in the attempted correlation.^ >22, 23
BR = K-JTT + K2F
BR = Kit?2 + <2V
The data from the phenolic series in Table 11 were evaluated by using
the seven possible correlations [Eq. (2) through (8)]. The results are
listed below, where r is the correlation coefficient and c is the molar
concentration at LCcg. The best correlations were obtained in Eq. (7) and
(8), which have both F and R dependency.
Log 1/c = 0.527 TT + 3.796 0.831 (9)
Log 1/c = 0.059 w2 + 0.371 TT + 3.851 0.835 (10)
Log 1/c = 0.339 a + 4.129 0.480 (11)
Log 1/c = 0.173 0 + 0.282 0 + 3.914 0.905 (12)
Log 1/c = 0.062 *2 + 0.095 IT + 0.364 0 + 3.613 0.965 (13)
Log 1/c = 0.500 TT + 0.453 F + 0.637 R + 3.731 0.978 (14)
Log 1/c = -0.028 Tr2 + 0.567 TT + 0.480 F + 0.607 R + 3.695 0.978 (15)
The error analysis showed that Eq. (14) represented the best correlation
as the T values observed for the coefficients of the Tr2 terms in Eq. (10),
(13) and (15) indicated that these parameters added little or nothing to
the correlation. The apparent improved correlation of Eq. (13) or (12)
is, therefore, not significant.
It must be emphasized that a correlation of this type is likely only
if a series is picked in which the mode of death remains constant, and at
best these correlations will only indicate trends. It can only be assumed
that extreme deviations correspond to a change in the mechanism of toxic
action.
Increasing halogen substitution (i.e., enhanced lipophilic nature)
in the phenol resulted in increased toxicity in agreement with the IT, F,
and R dependency. This observation is particularly significant in the
57
-------
current evaluation of the relative merits of chlorination and ozonation as
techniques for wastewater renovation and the previously observed incorpora-
tion of carbon-bound chlorine by a variety of phenolic systems under disin-
fection conditions.
Experimental
Three or more tests (four dose levels for each test) were run for each
compound under investigation. The data treatment was completely computerized
using least squares. However, for clarity it will be explained as if each
test were plotted by hand.
For each compound a graph was prepared plotting log percentage sur-
vivors vs. time in hours.^4 From this plot the percentage survivors for
each dose level at 48 hours could be determined. This procedure tends to
average the results and increase the repeatability factor involved with
biological testing. In addition, all values, from a 24-hour to a 96-hour
LC5Q can be calculated by using this one set of data. The 48-hour percent-
age survival figures were converted to probit values by using probit trans-
formation tables". These values were plotted vs. log molar concentration.
The log LC5Q molar concentration can then be found by observing the log
molar concentration value that corresponds to a probit value of 5. The pro-
bit values and log molar concentration values were introduced into a least
squares computer program which calculated the LC$Q and also gave the cor-
relation of the line to the data points.
This method for determining the 48-hours LC$Q value has the advantage
of a four-day observation period. The inconsistencies that arise when the
animals are counted only once (at 48 hours) are, therefore, averaged out,
and greater reproducibility of the data results. An example of this process
is illustrated for p_-cresol (Figures 4 and 5).
As a measure of the reliability of the screening procedure, a structure-
activity correlation was attempted for a series of phenols. Rather than
predict absolute values, we hoped to recognize trends within a given struc-
tural series, as has been done with correlations successfully carried out
in pharmaceutical drug design.14,26,27
REFERENCES
1. Bowden, F., water Poll. Cont. Federation J., 34, 1010 (1962).
2. Anderson, B. G., Sewage Works J., 1^, 1156 (1944).
3. Anderson, B. G., Sewage Works J.. 18, 82 (1946).
4. Anderson, B. G., Trans. Am. Fish. Soc.. Z8_, 96 (1950).
5. Biesinger, K. E. and G. M. Christensen, J. Fish. Res. Board Canada. 29_,
1691 (1972). —
58
-------
6. Anderson, B. G. in "Transactions Seminar on Biological Problems in
Water Pollution, 1959,"Robert A. Taft Sanitary Engineering Center,
Cincinnati, OH, Technical Rept. No. 60-3, 1960, pp. 94-95.
7. Burttschill, R. H., A. A. Rosen, F. M. Middleton and M. B. Ettinger,
J. Am. Water Works Assoc.. 51_, 205 (1959).
8. Eliasek, J. and A. Jungwirt, Coll. Czech!. Chem. Commun., J8_, 2163 (1963).
9. Nikolov, N. and V. Ilkov, Tezhka Prom. (Sofia), |, 26 (1963).
10. Sheychenko, M. 0., Yu. M. Kalinichik and A. N. Baranov'ska, Okr. Khim.
Zh... !£, 1105 (1963)5 Chem. Abstr., 60, 10390 (1964).
11. Schirst, 0., Fenol. Odpadni Vody, 26_ (1962); Chem. Abstr., 62_, 5066
(1965). — —
12. Musil, J., Z. Knotek, J. Chalupa and P. Schmidt, Sh. Vysoke Skoly Chem.
Techno!. Praze. Techno!. Vody. 8, 327 (1965); Chem. Abstr.. 65, 1g60,~T9.
L. A. Kul'skh, Yu. M. KalinichitT and A. N. Baranov'ska, KhimTTrom,
Nauk-Tech. Zb.. 21 (1962); Chem. Abstr.. 59, 4902 (1963).
13. Carlson, R. M., R. E. Carlson, H. L. Kopperman and R. Caple, Environ.
Sci. Techno!, g, 674 (1975).
14. Hansch, C., Ace. Chem. Res.. £, 232 (1969).
15. Hansch, C., R. M. Muir, T. Fujita, P. P. Maloney, F. Geiger and M.
Streick, J. Am. Chem. Soc., 85_. 2817 (1963).
16. Hansch, C. and T. Fujita, J. Am. Chem. Soc., 8£, 1616 (1964).
17. Fujita, T., J. Iwasa and C. Hansch, J. Am. Chem. Soc., jg, 1616 (1964).
18. Hansch, C., A. Leo, S. H. Unger, K. H. Kim, D. Nikaitani and E. J. Lien,
J. Med. Chem., V^, 1 (1968).
19. Jaffa, H. H., Chem. Rev., 53., 191 (1953).
20. Hansch, C. A. Lea, S. Unger, K. H. Kim. D. Nikaitani and E. J. Lien,
J. Med. Chem., 1_6, 1207 (1973); C. G. Swain and E. C. Lupton, Jr.,
J. Am. Chem. SocT, 90, 4328 (1973).
21. Hansch, C. in'Druq Design,"Vol. I, E. J. Ariens.ed., Academic Press
Inc., New York, N.Y., 1974.
22. Purcell, W. P., G. E. Bass and J. M. Clayton, "Strategy of Drug Design",
Wiley-Interscience, New York, N.Y., 1973.
23. Korolkovas, A., "Essentials of Molecular Pharmacology", Wiley-Inter-
science, New York, N.Y., 1970.
59
-------
24. (a) Frear, D. E. H. and J. E. Boyd, J. Econ. EntomoU 60., 1228 (1967).
(b) Frear, D. E. H. and N. S. Kawar, J. Econ. Entomol .~60_. 1236 (1967),
25. Finney, D. J. in "Statistical Methods in Biological Assay," 2nd ed.,
New York, N.Y., 1964, p. 630.
26. Hansch, C., A. Leo and D. Nikaitani, J. Org. Chem.. 37^ 3090 (1972).
27. Cammarata , A. and S. L. Yau, J. Med. Chem., 13_, 93 (1970).
PARTITION COEFFICIENTS via HIGH-PRESSURE LIQUID CHROMATOGRAPHY
Results and Discussion
The availability of a rapid and accurate technique for the determination
of partition coefficients (or their equivalent) became desirable when it was
observed that the partition coefficient between r^-octanol and water (PQ/W)
was dominant in the successful Hansch correlation of phenol toxicity to
aquatic species.' This concern has led to the development of a method that
uses a permanently bonded long-chain alky! packing in a high-pressure liquid
chromatographic system and subsequently relates the capacity factor k'k" =
(ts-to)/t0, i.e., the net retention time relative to the nonadsorbed time,
to the partition coefficient
It is well recognized that a separation (i.e., a difference in retention
volume) in "reverse-phase" chromatography depends upon the partitioning
characteristics of the solute between the mobile phase and the stationary
phase as represented by the value of the partition coefficient. To evaluate
the elution data in the current study, it was, therefore, necessary to con-
sider the relative number of moles of eluting solvent. However, the use
of mole percent resulted in a significant increase in the deviation from
linearity from that previously observed in the analysis of thin-layer
partition data2 when Rm [Rm = log (1/Rf-l)] was plotted against volume
percent.
For example, Figures 6 and 7 contain plots of log k' vs. volume percent-^
(sigma y = 0.012) and log k' vs. molar percent (sigma y = 0.035), respec-
tively, for a representative compound (£-chlorophenol ). It was subsequently
found that the linearity could be maintained and substantially extended
for all the compounds studied by plotting log (1/k' + 1) vs. mole percent
(e.g., p_-chlorophenol, Figure 7, sigma y - 0.009). This change is valid
as the correlations of Rm and k' are made over a range of percentages, and
the modification is, therefore, only from one empirical relationship to
another.
The correlations of log k' to log P and ir (IT = log Psubstituted ~
Tog ppare.nt) to K (K = log k'substituted - Io9 k'parent) for some phenols
and anilines are contained, along with their corresponding residuals, in
Tables 13 and 14. The coefficients obtained from the individual regression
analyses are found to be quite satisfactory. However, when the results
from the two families of compounds are combined, the correlation of log P
60
-------
to log k' decreases (r - 0.86). Although this is an acceptable value for
a regression analysis of this type, it indicates that P or k', or both,
reflect more than just lipophilic character.
PHENOLS:
log P = A log k' + C r = 0.96
IT = MK + C r = 0.96
ANILINES:
log P = A log k' + C r = 0.97
IT = MK + C r = 0.97
Related to these observations is the work of Collander3'^, which has
shown that although any two alcohol -water systems will provide a linear
relationship between log P values, it is not possible to extend the corre-
lation over a wide range of solvent types (alcohols, ester, ketones, halo-
genated hydrocarbons). The successful correlation in the present study,
therefore, indicates that either there is a good approximation of the solvent
forces in C-18 Bondapak/Acetone-H20 to those of alcohol/water systems or
that the chemical potential of either the lipophilic (H-|) or hydrophilic
(Hn) components of the solute remains constant. 5
log P =
2.3RT
In addition, although neither the octanol /water system nor the Bondapak C-18/
Acetone-H20 chromatographic system can be construed to be structurally
representative of a biological membrane, the somewhat comparable results
upon substitution of K for IT in a Hansch-type biological correlation6-9
(Table 12) indicate the predictive powers of k' and P in evaluating the
ability of an organic molecule to pass through biological tissue JO
log 1/c = M< + C r = 0.76
log 1/c = M< + C r = 0.68
Experimental
The separations were performed on 2-1/8" x 2' Bondapak C-18/Porasil B
columns that were mounted in a Waters Associates ALC 202 (refractive index
detector). The various mole percentages of distilled water and acetone
(MCP-ACS grade) were eluted at 24-26°C and a flow rate of 0.9-1.0 ml/mini.
61
-------
TABLE 12. COMPARISON OF OBSERVED AND CALCULATED BIOLOGICAL RESPONSES FOR
DAPHNIA MAGNA TOXICITY IN THE PRESENCE OF SUBSTITUTED PHENOLS.
Compound
Phenol
3-Methoxyphenol
4-Nitrophenol
4-Methyl phenol
2-Methyl phenol
2 -Chlorophenol
2,6 Dimethyl phenol
4-Chlorophenol
4-Bromophenol
Observed log
(1/c)
3.901
3.480
4.219
3.709
3.835
4.238
4.036
4.426
4.463
Calculated log
(1/c) from K
3.769
3.710
3.845
3.995
4.073
4.065
4.254
4.244
4.356
Calculated
(1/c) from
3.647
3.715
3.952
3.972
3.979
4.114
4.243
4.277
4.412
log
IT
REFERENCES
1. Kopperman, H. L., R. M. Carlson and R. Caple, Chem.-Biol. Interactions,
£, 245 (1974).
2. Biagi, G. L., A. M. Barbars, M. C, Guerra in "Biological Correlations -
The Hansch Approach", Advances in Chemistry, Series 114. American
Chemical Society, Washington, D. C., 1972, Chapter 5.
3. Karger, B. L., "Modern Practice of Liquid Chromatography," J. J. Kirk-
land, ed., Wiley-Interscience, New York, NY, 1971, Chapter 1.
4. Collander, R., Acta Physiol. Scand., 7_, 420 (1954).
5. Collander, R., Acta Chem. Scand., 5_, 774 (1954).
6. Green, J. and S. Marcinkiewicz, J. Chromatogr., 10, 35 (1963) .
7. Hansch, C., Accounts of Chem. Res.. 232 (1969).
8. Hansch, C., A. Leo and D. Nikaitani, J. Org. Chem., 37, 3090 (1972).
9. Cammarata, A. and S. L. Yarr, J. Med. Chem., 1^, 93 (1970).
10. Swain, C. G. and E. C. Lupton, J. Amer. Chem. Soc., 90, 4328 (1968).
11. Collander, R., Physiol. Plant.. 7, 420 (1954).
62
-------
TABLE 13. THE CORRELATION OF LOG k' TO LOG P AND TT TO K FOR SUBSTITUTED
ANILINES
Substituent
-H
3-OCH,
4-OCHj
2-CH,
3-NO2
4-CHj
3-CH,
2-NO,
4-a
2-a
4-Br
2,4-a
LogP
0.90
0.93
0.95
1.32
1.37
1.41
1.43
1.79
1.83
1.92
2.26
2.69
Logk'
-0.216
-0.145
-0.321
-0.004
0.029
0.021
0.004
0.083
0.182
0.210
0.262
0.585
Calc. log?
0.95
1.11
0.72
1.43
1.50
1.48
1.45
1.62
1.85
1.91
2.02
2.75
n
0.00
0.03
0.05
0.42
0.47
0.51
0.53
0.89
0.93
1.02
1.36
1.79
K
0.000
0.071
0.105
0.212
0.245
0.237
0.220
0.299
0.398
0.426
0.478
0.801
Calc. n
0.06
0.21
-0.18
0.53
0.60
0.58
0.55
0.72
0.95
1.01
1.12
1.85
TABLE 14. THE CORRELATION OF LOG k' TO LOG P AND TT TO K FOR SUBSTITUTED
PHENOLS
Substituent • L
-H
3-OCHj
4-NOj
4-CH3
2-CH3
2-CI
2,6-CHj
4-CI
4-Br
-OgP
.46
.56
.91
.94
.95
2.15
Z.34
2.39
2.59
Lop k'
-0.164
-0.213
-0.100
0.025
0.090
0.083
0.241
0.233
0.326
Calc. log P
1.61
1.52
1.73
1.97
2.09
2.08
2.38
2.37
2.54
0.00
0.10
0.45
0.48
0.49
0.69
0.88
0.93
1.13
0.000
-0.049
0.064
0.189
0.254
0.247
0.405
0.397
0.490
Calc. rr
0.15
0.06
0.27
0.51
0.63
0.62
0.92
0.91
1.08
63
-------
35
4O 45
Volume per cent woter
50
55
Figure 6. Plot of log k1 vs. volume per cent of water for p_-chlorophenol
O7SO
O7OO
0650
0600
0550
£ O500
s r
0400
0.350
0300
0250
0.2001
Sigma Y - 0 009
sigma Y'= O O35
X
0130
0060
0010
-OO8O
- -0150
-O.82O
Y'
JC
0360 o
-O.43O
-0.5OO
O.S7O
0620 0640 0660 0.660 07OO 0730 0740 0.760 0.78O O.BOO O.B2O 0640
Mole per cent water
0640
Figure 7. Plots of log [l/(k' + 1)] and k' vs. mole per cent of water for
p_-chlorophenol.
64
-------
BIOLOGICAL OXYGEN DEMAND OF CHLORINATED EFFLUENTS
Results and Discussion
The biological oxygen demand (BOD) reduction in chlorinated wastewater
is a well-recognized phenomenon."1^ However, the recent studies of Zaloum
and Murphy^ suggest that the dominant factor in BOD reduction is the signi-
ficant decrease in the microbial populations of the chlorinated wastewater
BOD test solutions when compared with the unchlorinated samples. The results
are also consistent with the conclusion that BOD reduction is not some
inherent character of the chemical process itself. Therefore, the observa-
tions were not associated with the possible oxidative conversion of the
soluble organics to some chlorinated progeny that might, in turn, prove to
be bioresistant or toxic, or both. However, this latter conclusion was
not reconciled with substantial previous evidence that, based upon the ini-
tial chlorine dose, approximately 1-2% of the available chlorine can be
accounted for as "second-order" chlororganics.3»4,5
Non-heterocyclic aromatics found in wastes subjected to chlorination
include phenols, benzoic acids, and. to a lesser extent, anilines, benzyl-
alcohols and polynuclear aromatics.3>6 Chlorinated examples of several of
these compound types have been found after chlorine disinfection.3 Among the
compounds present, phenolic material is not only the most ubiquitous, but
also the most vulnerable to chlorine incorporation over the entire range
of possible pH values.7 The presence of such chlorophenolic compounds may
indeed have an effect upon possible degradative processes, but proof would
depend upon an experimental design that would not only provide comparable
initial microbial populations, but also provide the opportunity to compare
any observation to a situation when no chlorination had occurred.
Results of BOD tests on dosed chlorinated phenols, benzoic acids and
anilines were evaluated against the parent compound (i.e., phenol, benzoic
acid and aniline) and against a control. The various compounds were com-
pared on a molar basis and, relative to the internal glucose-glutamic acid
standard, possessed the same initial microbial content. The results clearly
indicate that parent systems differ in their vulnerability during the test
period and that the "chloroproducts" have diminished degradability relative
to the parent. Moreover, the decrease in BOD relative to the internal
standard suggests that the "second-order" chlororganics in sufficiently
high concentration can have an adverse effect on the microbial community.
The results from the present investigation when compared to those of
Zaloum and Murphy demonstrate that the BOD test will not differentiate the
significant increase of the bioresistance of the chloroorganics when they
are present at sufficiently low levels. However, considering the apparent
toxic effects of some of the chlorinated materials at the ppm level, it would
be surprising if these materials are not contributing to the observed decrease
in the overall microbial population observed by these investigators. An over-
all summary of the investigation is provided in Tables 15-18.
65
-------
TABLE 15. SUMMARY OF BOD RESULTS FOR PHENOLS
CT>
Test #
I
II
2
6
1
1
1
2
6
1
2
6
1
X
X
X
X
X
X
X
X
X
X
X
10-6M <
10-6M
IO-SM
10-5M
10-3M
! *
o!
1 •
1 •
10'6M (0.
10'6M (0.
IO-SM
10-gM I
0.
0.
10'6M (0.
IO-SM (i.
19
56
94
29
29
19
56
94
33
98
63
ppm)
ppm)
PPm)
ppm)
ppm)
ppm)
ppm)
ppm)
ppm)
ppm)
ppm)
Phenol
(Days)
5 10
168
214
229
183
227
243
215
306
335
233
290
278
o-Chlorophenol £-Chlorophenol 2,4-Dichlorophenol
(Days) (Days) (Days)
20 5 10 20 5 10 20 5 10 20
284
290
335
165 207 229
187 223 245
269
290
273
135 159 166
154 156 173
151 149 147
1 X 10-5M (1.29 ppm)
150 168 170
Blank: Glucose-Glutamic Acid + seed = 176 (5 day); 237 (10 day); 232 (20 day)
-------
TABLE 15. SUMMARY OF BOD RESULTS FOR PHENOLS (CONTINUED)
(0-5 DAYS)
Sample1" 0 hr 24 hr 48 hr 72 hr 96 hr 120 hr
nig/ft mg/& mg/£ mg/£ mg/a mg/£
GGA* + seed 0 0 129 145 175 202
GGA + seed + phenol 0 0 175 199 241 274
GGA + seed + o-chlorophenol 0 5 118 140 170 203
GGA + seed + p-chlorophenol 0 12 128 165 194 212
GGA + seed + 2,4-dichlorophenol 0 10 129 129 174 200
Sample1" 0 hr 24 hr 48 hr 72 hr 96 hr 120 hr
GGA* + seed 0 0 134 181 235 264
GGA + seed + phenol 0 13 223 287 345 385
GGA + seed + cresol 0 10 209 274 359 384
GGA + seed + 2-chloro-5-methyl-
phenol 0 0 170 189 258 272
GGA + seed + 4-chloro-3-methyl-
phenol 0 0 0 40 144 161
f 1 X 10-5M in phenol or substituted phenol.
* GGA = Glucose-Glutamic acid; 5 ml of GGA per DO bottle was used. The BOD
values were calculated using 5 mis of GGA as the sample volume. One milli-
liter of settled (24 hrs) fresh sewage seed per one liter dilution water
was used. The incubator temperature was 20°C ±1°C throughout the incubation
period.
67
-------
TABLE 1 6. SUMMARY OF BOD RESULTS FOR BENZOIC ACID
Test # Benzoic Acid o-Chloro Benzoic Acid
1 5 10 5 10
2 X 10"6M (0.24 ppm) 258
6 X 10-6M (0.73 ppm) 254
1 X 10-5M (1.22 ppm) 246
2 X 10-6M (0.31 ppm)
6 X 10-6M (0.94 ppm)
1 X 10-5M. (1.57 ppm)
Blank: Glucose-Glutamic Acid +
238
244
246
seed = 154 (5
TABLE 17. SUMMARY OF BOD RESULTS
Test #
1 5
2 X 10-6M (0.19 ppm) 147
6 X 10-6M (0.56 ppm) 140
1 X 10-5M (0.93 ppm) 150
2 X 10-6M (1.28 ppm)
1 X 10-5M (12.8 ppm)
Blank: Glucose-Glutamic Acid
193 (20 day)
Aniline
10 20
179 178
153 171
161 141
+ seed = 155
142 166
142 152
129 156
day); 169 (10 day)
OF ANILINES
£-Chloro Aniline
5 10 20
131 140 120
44 140 114
(5 day); 161 (10 day);
1. Barnhart, E. L. and G. R. Campbell, "Effect of Chi orination on Selected
Organic Chemicals," U. S. Environmental Protection Agency, Washington,
D. C., Publication No. 12020 EXG03/72.
2. Zaloum, R. and K. L. Murphy, J. Water Poll. Contr. Fed., 46_, 2770 (1974).
3. Jolley, R. L., "Chlorination Effects on Organic Constituents in Ef-
fluents from Domestic Sanitary Sewage Treatment Plants," Ph.D. Disserta-
tion, University of Tennessee, Knoxville, TN, 1973.
4. Jolley, R. L., Environ. Lett.. 7, 321 (1974).
68
-------
5. Pitt, W. W., Jr., R. L. Jolley and C. D. Scott, Environ. Sci. Tech.. 9_,
1068 (1975).
6. Keith, L. H., Environ. Sci. Tech., 1_0, 555 (1976).
7. Carlson, R. M., R. E. Carlson, H. L. Kopperman and R. Caple, Environ.
Sci. Tech., 9., 674 (1975).
69
-------
APPENDIX A
SUMMARY OF LITERATURE ON DAPHNIA MAGNA TOXICITY
Acetic Acid
LD5024-47 ppm (l.SRW**)
max LDgct = 150 ppm* (3)
Acetone
LD5024-10 ppm (1,RDW)
LD5048-10 ppm (1,RDW)
max LOga = 9280 ppm (3)
Aldrifi
LD10050-29.2 ppb (2)
LD5024-30.0 ppb (4)
50-29.2 ppb (4)
LD5026-30.0 ppb (5)
Aluminum
LD5Q48-3.9 ppm (6.WOF)
Aluminum Sulfate
max LDga = 136 ppm (3)
Aluminum Ammonium Sulfate
max LD0o * 190 ppm (3)
Aluminum Potassium Sulfate
max LOga = 206 ppm (3)
Ammonium Chloride
LD5024-202 ppm (l.ULW)
LDso48-161 ppm (ULW)
LD5072-67 ppm (ULW)
LD5096-50 ppm (ULW)
LD50100-139 ppm (SRW)
max LDga = <134 ppm (3)
Ammonium Hydroxide
LD5025-60 ppm (1,SRW)
LD5Q50-32 ppm (1,SRW)
LD50100-20 ppm (1,SRW)
max LDga = <8.75 ppm (3)
Ammonium Sulfate
LD5Q25-423 ppm (1,SRW)
LD5Q50-433 ppm (l.SRW)
LD50100-292 ppm (1,SRW)
max LDga = <106 ppm (3)
Ammonium Sulfite
LD5025-299 ppm (l.SRW)
LD5050-273 ppm (SRW)
LD5gl00-203 ppm (SRW)
Aniline max LDga = 279 ppm (3)
LD5048-0.34 ppm (8)
o-Anisidine LD5Q48-4.71 ppm (8)
p-Anisidine LD5g48-0.73 ppm (8)
Anisole LD5048-140 ppm (7)
Anisonitrile LD5Q48-33 ppm (7)
Aramite LD5Q26-69.0 ppb (5)
Arsenic LD5048-7.4 ppm (6.WOF)
Atrazine LD5026-3.60 ppm (5)
* Maximum allowable for zero toxicity over infinite time
**SRW - Standard research water
ULW - University lake water
ROW - Reference dilution water
WOF - Without food in test
70
-------
Azinphosmethyl (Guthlon)
LD5026-180 ppt (5)
Barium LD5048-14.5 ppm (6.WOF)
Barium Chloride
max LDga = <83 ppm (3)
Benzene LD5g48-410 ppm (7)
Ben zoic Acid max LDga = 146 ppm (3)
Benzonitrile LD5048-250 ppm (7)
Benzyl alcohol LD5Q48-250 ppm (7)
P-Bromoaniline LD5048-0.20 ppm (8)
p-Bromoanisole LD5048-28 ppm (7)
p-Bromophenol LD5g48-5.9 ppm (7)
Butyric Acid LD5g48-61 ppm (1,SRW)
Cadmium LD5048-65 ppb (6.WOF)
Calcium LD5Q48-464 ppm (6)
Calcium Chloride
LD5025-3526 ppm (1 ,SRW)
LD5050-3005 ppm (l.SRW)
LD5024-1838 ppm (1 ,ULW)
LD5048-759 ppm (1,ULW)
LD5072-759 ppm (1 ,ULW)
LD501 00-649 ppm (l.ULW)
max LDga = 1332 ppm (3)
Captan LD5Q26-1.30 ppm (5)
Carbophenothion LD5026-9 ppt (5)
Carvone LDgg48-44 ppm (7)
2-Chloroaniline LD5048-0.61 ppm (8)
p-Chloroam'line LD5Q48-0.13 ppm (8)
Chlorobenzene LD5048-80 ppm (7)
Chi orobenzi late
LD-|0g50-1.4 PPm
LD5026-1.45 ppm (5)
o-Chlorophenol LD5048-7.6 ppm (7)
p-Chlorophenol LD5048-4.7 ppm (7)
Chlorthion LD5Q26-4.5 ppb (5)
Chromic Acid max LD0a = «0.6 (3)
Chromic Sulfate
LD5Q24-100 ppb (1.ULW)
LD5048-30 ppb (l.ULW)
Citric Acid max LDga = 153 ppm (3)
Cobalt LD5Q48-1.62 ppm (6)
Cobaltous Chloride
max LDga = «26 ppm (3)
Copper LD5Q48-60 ppb (6)
Coumaphos LD5o26-100 ppt (5)
Capric Chloride max LDga = 80 ppb (3)
Cupric Sulfate max LDga = 96 ppb (3)
Cyclethrin LD5026-55.0 ppb (5)
Dalapon (Acid) LD5g26-6.00 ppm (5)
ODD (IDE) LD5024-4.6 ppb (4)
DDT LD10050-1.4 ppb (2)
LD5024-4.4 ppb (4)
50-1.4 ppb (4)
LD5026-4.4 ppb (5)
Demeton LD5026-5.0 ppb (5)
Diazinon
LDigg50-4.3 ppb (2)
LD5050-4.3 ppb (4)
Dicapthon LD5Q26-4.1 ppb (5)
Dichlone LD5g26-26.0 ppb (5)
2.4-Dichloroaniline LD5g48-2.93 ppm (8)
2.5-Dichloroaniline LD5g48-2.94 ppm (8)
3.4-Dichloroaniline LD5g48-0.32 ppm (8)
2,4-Dich1oro-6-(o-Chloroaniline)-5-
Triazine LDc;n26-490 ppb (5)
2,4-Dichlorophenol LD5048-2.6 ppm (7)
Dicofol LD5026-390 ppb (5)
Pi eldrin
LD10050-330 ppb (2)
71
-------
LD5o24-740 ppb (4)
50-330 ppb (4)
LD5026-740 ppb (5)
Pi Ian LD5026-21.0 ppb (5)
Dimethoate LD5026-2.50 ppm (5)
m-Dimethoxybenzene
LD5048-98 ppm (7)
2,6-Dimethylphenol
LD5048-11 ppm (7)
Dimite LD5Q26-290 ppb (5)
2.4-Dinitroaniline
LD5048-10.7 ppm (8)
2,4-Dinitrophenol
LD5048-4.8 ppm (7)
Dloxathion LD5026-330 ppt (5)
Dipterex LD5050-120 ppt (6)
Dodlne LD5026-58.0 ppb (5)
Doweo 109 LD5026-400 ppb (5)
Endosulfon LD5Q26-240 ppb (5)
Endrin LD10050-352 ppb (2)
LD5o26-900 ppb (5)
LD5024-900 ppb (4)
50-352 ppb (4)
EPN
Phenylphosphonothioic acid o-ethyl
o-p-nitrophenyl
LD10050-100 ppt (2)
LD5026-nO ppt (5)
Ethion LD5026-72 ppt (5)
Ethyl alcohol
max LDga = 18400 ppm (3)
Ethyl N,N-d1propy1-thiocarbamate
LD5026-1.70 ppm (5)
Ethyl parathlon
LD5024-800 ppt (4)
50-800 ppt
Ferbam LD5026-85.0 ppb (5)
Ferric Chloride
LD5025-36 ppm (1,SRW)
LD5050-21 ppm (1 ,SRW)
LD50100-15 ppm (l.SRW)
max LDgcx = 130 ppm (3)
Ferrous Sulfate
max LDga = <152 ppm (3)
Formaldehyde
LD5024-<100<1,000 ppm (1 ,RDW)
Glyoden LD5026-300 ppb (5)
Guaiacol LD5048-26 ppm (7)
Heptachlor
LD10050-57.7 ppb (2)
LD5024-52.0 ppb (4)
50-57.7 ppb
LD5026-52.0 ppb (5)
Heptachlor Epoxide
LD5026-120 ppb (5)
Hydrochloric Acid
max LD0ct = 62 ppm (3)
Iron LD5Q48-9.6 ppm (6)
Iso-amyl Alcohol
max LDgct = 881 ppm (3)
Isolan LD5026-12.5 ppb (5)
Isopulegal LD5Q48-74 ppm (7)
Lactic Acid max LDga = 243 ppm (3)
Lead LD5048-450 ppb (6)
Limonene LD5Q48-6.1 ppm (7)
Lindane LD5050-1.100 ppm (4)
LD5Q26-1.25 ppm (5)
Magnesium LD5048-322 ppm (6)
Magnesium Chloride
LD5025-3391 ppm (l.SRW)
72
-------
LD5o50-3699 ppm (SRW)
LD50100-3484 ppm (SRW)
Magnesium Sulfate
LD5024-963 ppm (1,ULW)
LD5048-929 ppm (ULW)
LD5072-861 ppm (ULW)
LD5096-788 ppm (ULW)
LD5096-3803 ppm (SRW)
Malathion LD10o50-900 ppt (2)
LD5Q24-900 ppt (4)
50-900 ppt
LD5026-900 ppt (5)
Manganese LD5048-9.8 ppm (6.WOF)
Mercury LD5Q48-5 ppb (6.WOF)
p-Methoxybenzyl alcohol
LD5048-130 ppm (7)
Methoxychlor LD-]g050-3.6 PPb (2)
LD5024-3.7 ppb (4)
50-3.6 ppb
LD5026-3.7 ppb (5)
m-Methoxyphenol LD5g48-41 ppm (7)
p-Methoxyphenol LDso48-2.6 ppm (7)
Methyl Alcohol
max LDga = 32000 ppm (3)
Methyl parathion
LD5o26-4.8 ppb (5)
2-Methylphenol LD5048-15 ppm (7)
3-Methylphenol LD5048-28 ppm (7)
4-Methyl phenol LD5Q48-22 ppm (7)
Naled LD5026-360 ppt (5)
Nemacide (0-2,4-dichlorophenyl)
(0,0-diethyl phosphorothioate)
LD5026-1.1 ppb (5)
Nickel LD5048-1.12 ppm (6)
Nitric Acid max LOga = 107 ppm (3)
o-Nitroaniline LD5Q48-12.0 ppm (8)
m-Nitroaniline LD5o48-2.71 ppm (8)
p-Nitroaniline LD5Q48-13.8 ppm (8)
p-Nitroanisole LD5Q48-12 ppm (7)
p-Nitrophenol LD5048-8.2 ppm (7)
Oxalic Acid max LOga = 95 ppm (3)
Parathion LD10Q50-800 ppt (2)
LD5026-800 ppt (5)
Perthane LDso26-9.4 ppb (5)
Phenol D. magna
LD5024-100 ppm (l.RDW)
LD5048-100 ppm (1,RDW)
D. magna (young)
LD5025-17 ppm (1 ,SRW)
LD5050-7 ppm (1,SRW)
D. magna (adult)
LD5025-61 ppm (l.SRW)
LD5021 ppm (l.SRW)
max LDga = 94 ppm (3)
LD5048-12 ppm (7)
p-Phenylphenol LD5048-3.6 ppm (7)
Phorate LD5026-2.2 ppb (5)
Phosphamidon
LD5050-12.5 ppb (4)
LD5026-4.0 ppb (5)
Phostex LD5o26-16.1 ppb (5)
Potassium LD5048-166 ppm
Potassium Chloride
LD50100-679 ppm (1,SRW)
LD5024-343 ppm (1 ,ULW)
48-337 ppm (l.ULW)
72-117 ppm (1,ULW)
96-29 ppm (l.ULW)
max LDQa = 373 ppm (3)
73
-------
Potassium Cyanide
LD5024-2 ppm (1,ULW)
48-2 ppm (l.ULW)
72-700 ppb (1,ULW)
96-400 ppb (1,ULW)
Potassium Dichromate
LD50100-400 ppb (l.SRW)
max LD0a = «600 ppb (3)
Potassium Fem'cyanlde
LD5024-905 ppm (l.ULW)
48-549 ppm (1,ULW)
72-600 ppb (1,ULW)
96-100 ppb (l.ULW)
Potassium Nitrate
LD5024-490 ppm (1,ULW)
48-490 ppm (1,ULW)
72-226 ppm (1,ULW)
96-39 ppm (l.ULW)
LD50100-900 ppm (1,SRW)
Potassium Permanganate
max LD0a = 630 ppb (3)
Prometone LD5026-35.0 ppm (5)
Propionic Acid
LD5048-50 ppm (l.SRW)
Pyridine LD5o24-2,114 ppm (l.RDW)
48-944 ppm (1)
Ronnel LD5026-1.8 ppb (5)
Sodium LD5048-1820 ppm (6)
Sodium Anthraguinone
alpha-sulfonate
LD5024-186 ppm (1,ULW)
48-186 ppm (l.ULW)
72-186 ppm (1,ULW)
96-50 ppm (l.ULW)
LD5Q100-12 ppm (SRW)
Sodium Arsenate
max LDga = 31 ppm (3)
Sodium Bicarbonate
max LDga = 4200 ppm (3)
Sodium Bisulfite
D. magna - young
LD5025-116 ppm (l.SRW)
50-81 ppm (l.SRW)
D. magna - adult
LD5096-102 ppm (1 ,SRW)
Sodium Bisulfite
D. magna - adult
LD5Q24-171 ppm (1 ,ULW)
48-119 ppm (l.ULW)
72-97 ppm (l.ULW)
96-82 ppm (l.ULW)
Sodium p-bromobenzene sulfonate
LD5024-2347 ppm (l.ULW)
48-1943 ppm (1 ,ULW)
72-971 ppm (l.ULW)
96-809 ppm (l.ULW)
LD50100-523 ppm (l.SRW)
Sodium Butyl Sulfonate
LD5024-8,000 ppm (l.ULW)
48-8,000 ppm (l.ULW)
72-5,400 ppm (1,ULW)
96-2,700 ppm (l.ULW)
Sodium Carbonate
LD5024-347 ppm (1,ULW)
48-265 ppm (1,ULW)
LD5025-607 ppm (l.SRW)
48-565 ppm (1,SRW)
96-524 ppm (l.SRW)
max LDga = 424 ppm (3)
74
-------
Sodium Chloride
LD5025-6447 ppm (l.SRW)
50-5874 ppm (1,SRW)
100-3114 ppm (l.SRW)
LD5024-3412 ppm (l.ULW)
48-3310 ppm (1,ULW)
max LDga = 6143 ppm (3)
Sodium p-chlorobenzene sulfonate
LD5Q24-8600 ppm (l.ULW)
48-7659 ppm (1,ULW)
72-3964 ppm (l.ULW)
96-2150 ppm (l.ULW)
LDsol00-2394 ppm (1 ,SRW)
Sodium 2-chlorotoluene-5-su1fonate
D. magna - young
LD5Q25-800 ppb (l.SRW)
50-600 ppb (l.SRW)
100-400 ppb (l.SRW)
D. magna - adult
LD5Q25-3.3 ppm (1,SRW)
50-.1.3 ppm (1,SRW)
Sodium 2,5-dichlorobenzene sul-
fonate
LD5024-4931 ppm (1,ULW)
48-4931 ppm (1,ULW)
72-2490 ppm (1,ULW)
96-938 ppm (1,ULW)
LD50100-1468 ppm (1,SRW)
Sodium Pichromate
LD5024-22 ppm (1,ULW)
48-10 ppm (l.ULW)
Sodium Hydroxide
max LDQa = 240 (3)
Sodium Methyldlthlocarbamate
LD5026-330 ppb (5)
Sodium mono-hydrogen phosphate
LD5025-1154 ppm (1,SRW)
50-1089 ppm (l.SRW)
100-426 ppm (l.SRW)
Sodium Nitrate
LD5024-5980 ppm (1,ULW)
48-3581 ppm (l.ULW)
72-2125 ppm (1,ULW)
96-665 ppm (1,ULW)
LD5096-665 ppm (l.SRW)
max LDga = 8500 ppm (3)
Sodium m-nitrobenzene sulfonate
LD5024-8665 ppm (l.ULW)
48-9665 ppm (1 ,ULW)
72-6017 ppm (l.ULW)
96-5067 ppm (1 ,ULW)
LD50100-2235 ppm (1,SRW)
Sodium 4-nitrochloro-benzene-2-su1
fonate
LD5024-4698 ppm (l.ULW)
48-3483 ppm (1 ,ULW)
72-948 ppm (l.ULW)
96-948 ppm (1,ULW)
LD5Q96-1474 ppm (1,SRW)
Sodium p-phenolsulfonate
LD5Q24-13510 ppm (1 ,ULW
48-13510 ppm (l.ULW)
72-3594 ppm (1,ULW)
96-1471 ppm (l.ULW)
Sodium Phosphate
LD5Q25-237 ppm (l.SRW)
50-177 ppm (1,SRW)
75
-------
100-126 ppm (l.SRW)
Sodium Pyrophosphate
LD5024-433 ppm (l.ULW)
48-391 ppm (l.ULW)
Sodium Sillca-te
LD5024-575 ppm (1,ULW)
48-494 ppm (1,ULW)
72-413 ppm (l.ULW)
96-216 ppm (l.ULW)
LD5096-247 ppm (1,SRW)
Sodium Sulfate
D. magna
LD5024-8384 ppm (l.ULW)
48-2564 ppm (1 ,ULW)
72-725 ppm (l.ULW)
96-630 ppm (l.ULW)
D. magna - adult
LD5096-4547 ppm (l.SRW)
D. magna - young
LD5024-6800 ppm (1,SRW)
48-6100 ppm (l.SRW)
max LDQa = 7105 ppm (3)
Sodium Sulfide
LD5025-16 ppm (l.SRW)
50-13 ppm (l.SRW)
100-9 ppm (l.SRW)
Sodium Sulfite
LD5Q25-299 ppm (1,SRW)
50-273 ppm (1,SRW)
100-203 ppm (l.SRW)
max LDQa=3784 ppm (3)
Sodium Thlosulfate
LD5025-2245 ppm (l.SRW)
50-1334 ppm (l.SRW)
100-805 ppm (1,SRW)
Strontium LDso48-125 ppm (6,WOF)
Sulfuric Acid max LDga = 88 ppm (3)
Sulphenone LDso26-210 ppm (5)
Tanm'c Acid max LDga = «26 ppm (3)
Tartaric Acid
max LDga = 135 ppm (3)
TDE LD5Q26-4.5 ppb (5)
a-Terpineol LD5Q48-120 ppm (7)
Thanite LD5026-900 ppb (5)
Thiram LD5Q26-1.30 ppm (5)
Tin LD5Q48-55 ppm (6)
m-Toluidine LD5048-0.18 ppm (8)
o-Toluidine LDso48-3.52 ppm (8)
Toxaphene LD5o26-94.0 ppb (5)
2,4,6-Tri bromephenol
LD5048-1.3 ppm (8)
Trichlorfon LD5026-120 ppt (5)
Valeric Acid LD5048-45 ppm (1 ,SRW)
Xylene LD5024-<100<1,000 ppm (l.RDW)
Zinc LDse48-280 ppb (6)
Zinc Sulfate max LD0a = <48 ppm (3)
Ziram LD5026-16.0 ppb (5)
Zineb LD5026-200 ppb (5)
76
-------
BIBLIOGRAPHY
1. Dowden, B. F. and H. J. Bennett, Journal of the Water Pollution Control
Federation. 3_7_, 9, 1308-16 (1965).
2. Anderson, B. 6., "The Toxicity of Organic Insecticides to Daphnia," in
Transactions, 1959 seminar of the Robert A. Taft Sanitary Engineering
Center, Cincinnati, Ohio, Technical Report No. 60-3, 1960.
3. Anderson, B. G., Journal of the Water Pollution Control Federation, 16,
6, 1156-1165 (1944T —
4. Sanders, H. 0. and 0. B. Cope, Transactions of the American Fisheries
Society, £|f 165-169 (1966).
5. Frear, D. E. H. and J. E. Boyd, Journal of Economic Entomology, 60_, 5,
1228-36 (1967). —
6. Biesinger, K. E. and G. M. Christensen, Journal of the Fisheries Re-
search Board of Canada. 29_, 1691-1700 (1972).
7. Carlson, R. M. and R. Caple, unpublished data, University of Minnesota,
Duluth, Minnesota.
77
-------
APPENDIX B
COMPUTER PROGRAM FOR COMPUTATION OF LD50
PROGRAM BIOASSAY
The objective of this program is to allow the computer to calculate
the 1050 (or any other specified biological response).
This program assumes data has been collected periodically over some
given time period. (3 or more points). Therefore, the first step is to
perform a least squares computation on this data and calculate the required
information which will be needed for the second step.
In our case we counted survivors (96 hr statics using Daphm'a magna)
every 24 hours for 96 hrs, carried out a weighted least squares treatment
on the data and from that calculated the expected percent survivors at
48 hours.
The second step in the program incorporates the percent survivors
at 48-hour information into another least squares treatment (unweighted)
which in turn yields the desired LCsg information. In this calculation a
least squares treatment is carried out on a plot of molar concentration
versus probit percent survivors at 48 hours.
As stated this program uses least squares treatment to analyze the
data. The main program has at its disposal two subroutines, one which is
an unweighted least squares program and the other is a weighted least
squares program.
The weighted program was used in the first step giving the largest
weight to the initial point (time zero), while the last point (time final)
was given the smallest weight. The reasoning being that at Tg we assumed
all animals to be healthy, and at any time after that we can nave doubts
about the survivors' health and at the end of the test, we are dealing
with the super hardy fraction which is not a true representation of the
average animal population.
Directions for punching and arranging data cards
The program is designed to accept data in the following format. The
first 10 data cards (10F5.3) are probit values which must be read into
the computer each time. These values were taken from the literature.
Next, data involving one compound at a time is fed into the computer
as follows:
-------
FIRST CARD
1234
1 0
M.W.
15—»38 column spacing on card
compound name
A B C
A. The number of dose levels tested punched in card (14 format).
This example has 10 dose levels.
B. The molecular weight (M. W.) is punched in next (F 10.4 format).
C. The next piece of information on this card is the compound name,
spaces 15 to 38 are used for this.
The next card refers to the first dose level tested. This card has
the information about the number of data points (A; 5 in this example)
and the molar concentration for the dose (B).
SECOND CARD
1234
5—»14
molar cone
B
This card stipulates that there are 5 data points; therefore, the next
5 cards contain the bioassay data which was collected. An example for one
of these cards is shown. Each card contains time, percent survivors and
the weight factor which should be given that particular point (all are F10.4
format).
1 >10 11—>20 21—^30
TIME % SURVIVORS WEIGHT FACTOR
At this point all the points for one dose level have been entered.
This process is repeated for each dose level by punching a card analogous
to the second card described above. After all the dose levels and their
corresponding data points have been read in, the next compound is entered
using a card analogous to the first card described above and the whole
process repeated for it. This is repeated until all compounds and data
have been entered.
The last card in the data deck must have a zero punched in space 4.
This will terminate the program.
79
-------
00
o
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
PROGRAM BIOASSflY
THIS PROGRAM IS DESIGNED TO ANALYZF BIOASSAY DATA AND PRINT OUT THE
RESULTANT LC5C'S IN THE FOLLOWING MANNER FOR A MAXIMUM OF 30 COMPOUNDS
NAME NO. OF DOSE LC50 CORRELATION SIGMA
LEVELS LOG 1/C PPM (LOG 1/C)
0-CRESOL 7 3.798 17.22 ,9
-------
oo
C ALL DATA READ IN IS WRITTEN OUT AGAIN IMMEDIATELY AFTERWARD FOR CHECKING
C
C J = 0 CALL EXIT - PROGRAM TERMINATED (PLACE 2ERO IN COLUMN «4
C OF LAST CARD IN DATA DECK
C
DIMENSION PR OBIT (IOC) ,AMW(30) , PPM (30, 30) , ANA ME (
-------
00 20 N s l,K
20 FISH(N) = ALOG10(OAFNIA(N))
C
C MASK = W SUSROLTINE PRINT OUT
C
C MASK = 4HMASK NO SUCROUTINE PRINT, OUT
C
MASK = <*H
WRITE(61.10i«)
C
C THIS SUBROUTINE USES A WEIGHTED LEAST SQUARES. THE WEIGHT FACTOR IS
C 1/(WT SQUARED) THEREFORE A SMALL NUMBER FOR WT RESULTS IN A LARGER
C EMPHASIS BEING PLACED ON THAT DATA.
C
CALL WE IGHT (K, T IME , FI SH, VIT , CONST, SLOPE, MASK fR»S)
C
C THIS STATEMENT STORLS THE STANDARD DEVIATION IN WT2 AND CAN BE USED
C IN LATER CALCULATIONS AS A WEIGHT FACTOR
C
ro WT2(M) = S
C
C THE NEXT THREE STATEMENTS CALCULATE THE FROBIT VALUE TO BE USED
c IN THE SECOND PLOT. A ROUND OFF COMMAND is USED HFRE, BECAUSE ONLY
C PROOIT VALUES FOR WHOLE NUMBERS WHERE SUPPLIED INITIALLY
C
YP = l»8.0*SLOFE * CONST
IP = IFIXUO**YP + UE)
PROQ(M.I) a PROUITdP)
C
C LOG 1/C IS CALCULATED AT THIS POINT
C
30 3R(M,I) = ALOGlti UAhrt (I)/PPM(M,I))*10**3)
MASK = ^H
WRITE(61,107) (ANAME (II,I) , II = !,«•»
C
C THIS SUBROUTINE USES UNWEIGHTED LEAST SQUARES TREATMENT
C
CALL LINEFIT(J,I»BR,PROBtA,B,SIGMAY,MASK)
-------
ELC50(I) = (5 - A)/£
APPrt(I) = AMMI)*10*»<3 - ELC50(I)>
SIGMAX(I) * SIGMAY/6
DO 35 M = 1,J
U(M) = BR(M,I)
35 V(M) = PROB(M.I)
MASK = *»H
C
C THIS SUBROUTINE USES WEIGHTED LEAST SQUARES TREATMENT
C
c SAME: DATA USED - THIS TIME WEIGHT FACTORS INCLUDED
c
c
CALL WEIGHT
-------
I END = I
GO TO 1
199 WRITE(61,109)
WRITE(61,105)
00 50 I a l.IENO
50 WRITE(61,106) (ANAME ( II , I) , II = 1,U), JDOSE ( I) ,ELC50 ( I) » APPM C I) •
$ RR(I), SIGhTX(I)
100 FORMATdOF5.3)
101 FORMATC I't.FlO.^.'tAe)
102 FORMATdi»,F10.i»)
103 FORMAT(3F10.U
105 FORMAKSX^l'iH COMPOUND NAME,6X,7H NO. OF.13X.5H LC50,10X,12H CORRE
$LATION,3X,6h SI&MA/L6X.5H OOSE,«*3X,8H LOG 1/C/25X.7H LEVELS, 6X,8H
JLOG 1/C,6X,*4H PPM//)
106 FORMAT(1HO,«4/»6,3X, I2,3X,5(5XfF7.3»
107 FORMAT(1HO,««A6,1EH FRORIT PLOT...//)
109 FORMAT(1H1,63H CALCULATED LETHAL CONCENTRATION (LOG 1/C, C = MOLA
$R CONCENTRATICN)/««8H FOR 50 PERCENT'tF THE TEST A.NIPALS AT *»8 HOUR
$S.)
200 FORMAT(1H1,I<4,F10.'»,'»A6)
201 FORMAT(1HO,I<«,F10.<* /)
202 FORMATdH ,3F10. A ,B, MASK, R, SIGMA)
DIMENSION U130),V(30), WT130), R(30), YY(30)
XN = FLOAT(N)
SUMX = 0.0
SUMY = 0.0
SUMDELXY = O.C
SUMDELXR = 0.0
SQELB? = O.,0
SOELA2 = 0.0
-------
W DO 3 I =1,N
SUMRSQ = 0.0
SUMri = 0.0
00 1 I = l.N
W = 1.0/
-------
99 FORMATIlHO,26HrME BEST LINE FIT IS 2X,F10.6,8H » X + ,
+F12.6//32H THE UNCERTAINTY IN THE SLOPE IS,3X,F10.6,
+ 2 )
101 FORMATIiHO,10X,lCHSIGKA Y = ,F10.6/>
105 FORMATdH .I5,2X,2flH POINTS AND X RUNNING FROM ,F10.6,*»H TO *
+F10.6)
106 FORMATdH ,7X,2M X , 12 X , 1HY ,12X,<»HF (X) , 9X,<»H WT ,9X, 1 OHRESIOUALS
END
SUBROUTINE LINE FIT ( N, KN,X ,Y, A ,EUSIGMAY ,HASK)
DIMENSION X(30,JO),Y(30,30)
XN= FLOAT(N)
SUMX= 0.0
SUMY= 0.0
SUHDELXY = 0.0
SUMOELX2 = 0.0
« SDELB2= 0.0
SDELA2= 0.0
RESI02= 0.0
DO 1 1=1,N
SUMX = SUMX * X(I,NN)
SUMY = SUMY * Y(I,NN)
XBAR = SUMX/ XN
YBAR = SUMY/ XN
DO 2 I = 1,N
OELX = X(I,NN) - XBAR
DELXY = (Y(I,M) - YBAR)*DELX
DELX2 = DELX ** 2
SUMDELXY = SUMOcLXY * OELXY
SUHDELX2 = SUKOELX2 •«• OELX2
B = SUMOELXY / SUMDELX2
A = YBAR - B * XBAR.
-------
DO 3 1= 1,N
IFIABSIX(I*NN)).LT. .1E-Q5) GO TO 3
SOELB2 =<(Y(I,NN» - A)/X(I,NN) - B) **2 + SDELB2
3 SOELA2 =(Y(I,NN) - E*X(I,NN) - A)**2 + SDELA2
OELTAB = SORTISUELB2 / XN)
DELTAA = SQRT(SDELA2 / XN)
IF ( MASK .EG. 4HMASK ) RETURN
WRITF.161,105) N, X(1,NN), X(N,NN)
MRITE(61«99) E , A ,DELTAB ,DELTA A
PRINT 106
DO i* 1= 1, N
F = 8*X(I.NN) + A
RESIO = Y(I.NN) - F
WRITE(61,100) X(I,NN)« Y(I.NN), F, RESID
i* RF.SI02 ~ RESID ** 2 * RESID2
SIGMAY - SGRT(RESI02/ XN)
PRINT 101.SIGKAY
RETIRN
99 FORMAT(1H!J,26HTHE BFST LINE FIT IS. ,2X,F10.6,8H * X * ,
+F12.6//32H THE UNCERTAINTY IN THE SLOPE IS,3X,F10.6,
+ 2^H AND IK THE INTERCEPT IS ,3X ,F10,6//>
100 FORMAT( 1H .
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-77-066
3. RECIPIENT'S ACCESSION>NO.
4. TITLE AND SUBTITLE
Chemical/Bioloqical Implications of Using Chlorine
and Ozone for Disinfection
5. REPORT DATE
June 1977 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Robert M. Carlson and Ronald Caple
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Department of Chemistry
University of Minnesota-Duluth
Duluth, Minnesota 55812
10. PROGRAM ELEMENT NO.
1BA608
11. CONTRACT/GRANT NO.
Grant R-800675
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory-Duluth, MN
Office of Research and Development
U.S. Environmental Protection Agency
Duluth, Minnesota 55804
13. TYPE OF REPORT AND PERIOD COVERED
Final Project 1972-76
14. SPONSORING AGENCY CODE
EPA/600/03
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Chlorine is readily incorporated into a variety of organic materials known
to be present in water subjected to chlorine-renovation procedures. The observed
products can be predicted on the basis of commonly used mechanistic considerations.
The aqueous ozonation studies confirm that mechanistic considerations developed
in non-aqueous cases can be applied to the prediction of products from ozone
addition to dilute solutions of unsaturated organics in water.
The dominant feature in the observed toxicity of phenols to Daphnia magna
was the lipophilic nature of the compound as represented by the partition
coefficient. The partition coefficient of a compound has been shown as part of
this overall study to be readily obtained from its retention properties on a
"reverse-phase" HPLC column.
The effects of chlorination on biological oxygen demand (BOD) were examined
by comparing the BOD requirements of a sample containing a given parent system vs_
that of its chlorinated progeny. The results indicate that the chlorinated
material is generally degraded less than the parent and that the lowered BOD values
appear, at least for phenols, to be associated with the increased toxicity of the
chlorinated material to the degrading organism.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Chlorination
Ozonization
Disinfection
Water Treatment
Bioassay
Toxicity
Contaminants
Chemical analysis
Chemical composition
Biochemical oxygen
demand
Chemical reactions
Chemical structure-
toxicity correlations
06/F
06/T
07/C
8. DISTRIBUTION STATEMENT
RELEASE TO THE PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
21. NO. OF PAGES
96
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
88
:>U.S. GOVERNMENT PRINTING OfFlCi 1977-757-056/6^23 Region No. 5-I I
------- | |