United States
Environmental Protection
Agency
Health Effects
Research Laboratory
Research Triangle Park, NC 27711
Research and Development
EPA/600/S1 -89/006 July 1989
Project Summary
The Isolation and
Identification of Electrophilic
Mutagens Produced During
Chlorine Disinfection
Robert M. Carlson
The chlorination of organic
materials present in natural waters
and in wastewaters generates many
direct acting mutagenic and
potentially carcinogenic products.
The reaction of nucleophilic reagents
with the unidentified mutagenic
electrophiles present in disinfected
water and with a number of known
electrophilic compounds as a means
of generating stable additioin
products that would aid in the
isolation, chromatographic sepa-
ration, and in the identification of the
mutagens via mass spectrometry was
investigated. Of the nucleophiles
investigated for this purpose
diethyldithiocarbamate proved to be
the most suitable. It was found to
react with most of the electrophiles,
but the mass spectra of the resulting
products while providing unique ions
suitable for selective ion monitoring,
were not suitable for structure
determination. Nucleophilic
functionalities bonded to solid
supports were also investigated for
the purpose of selectively isolating
electrophilic mutagens from aqueous
samples and while several were
found that would remove the
electrophiles, no suitable means of
removing them from the supports
was found.
This Project Summary was
developed by EPA's Health Effects
Research Laboratory, Research
Triangle Park, NC, to announce key
findings of the research project that is
fully documented in a separate report
of the same title (see Project Report
ordering information at back).
Introduction
Chlorination of the naturally occurring
organics in water generates a variety of
new products. Based upon results
obtained from biological assays, it has
been determined that some of these
products are mutagenie. Many of the
compounds arising from the chlorination
process require no enzymatic activation
to exhibit their mutagenic activity. These
direct acting mutagens are relatively
unstable compounds that will attack a
nucleophilic reactant that possesses a
readily available source of electron
density (e.g., non-bonded electrons, pi
systems, anions). Such electrophiles as
alkylhalides, epoxides, low molecular
weight aldehydes, and halogenated
carbonyl systems are representative of
this electrophilic reactant type that form
during chlorination and that may interact
with a nucleophile to undergo such
reactions as substitution. In the situation
where the nucleophile is a molecule of
significant biological importance, the
properties of the newly formed product
are likely to be adverse.
It was known from earlier
investigations that an'external reactive
nucleophilic reagent such as thiosulfate
eliminated the mutagenic activity
detectable in chlorinated wastewater with
the Ames Salmonella assay. The
presumed mode of reaction was through
reduction and nucleophilic addition. The
results of preliminary studies indicated
that it may be possible to develop an
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analytical methodology in which
nucleophilic reagents would be reacted
with the electrophiles in the sample to
form products that would exhibit
distinctive properties that would aid in
their isolation and identification.
Moreover, such an analytical scheme
using nucleophilic tags would render the
olsctrophilic mutagens harmless to
laboratory personnel and also make them
more amenable to manipulation.
The primary mode of compound
identification currently used in these
types of studies is gas chromatog-
raphy/mass spectrometry (GC/MS).
Therefore the labeled compounds must
be sufficiently volatile to allow a GC
separation without a significant
probability of decomposition. It was also
anticipated that physical or chemical
properties of the covalently bound label
(e.g., acidic or basic functionality,
lipophilicity, fluorescence, radioactivity or
electrochemical properties) would
incorporate distinctive detection
characteristics that would aid in the
isolation of enriched fractions from the
remainder of the complex mixture. The
nucleophilic substances evaluated are
listed in Table 1.
Another method for the selective
isolation of electrophilic substances from
water that was investigated was an
insoluble polymeric matrix into which had
been incorporated a removable
nucleophilic label. The change in physical
and chemical state (e.g., liquid to solid,
reactive to non-reactive) would allow a
ready separation from the complex matrix
remaining dissolved in the water or
merely adsorbed to the polymer surface.
Moreover, it was hoped that this
procedure would trap the more reactive
mutagenic materials that might otherwise
elude discovery. Cleavage of the
modified electrophiles from the polymer
will be accomplished by mild chemical
procedures to give a cohort of organic
materials that could be identified by
conventional mass spectral techniques.
The polymeric reagents evaluated are
listed in Table 2.
Results
The underlying concept, that reactive
mutagenic electrophiles present in
mixtures would react with nucleophilic
probes resulting in decreased biological
activity is strongly supported by this
work. Of the nucleophiles tested,
diethyldithiocarbamate (DEDTC) affected
the most rapid reduction-ofmutagenicity. -
Cyanide and nitrogen bases including N-
methyl-2-mercaptoimidazole, 2-
mercaptopyridine, 2-mercaptopyrimidine
and thiazole caused a much less
dramatic decrease.
DEDTC emerged as the prime
candidate for labeling of aqueous
electrophilic mutagens. Positive features
of DEDTC included: 1) the label high
nucleophilic reactivity, 2) mass spectral
patterns evolved (viz. 116/148/149) that
were very characteristic of labeled
dithiocarbamate, 3) the derivatives had a
relatively high (15,000) UV extinction
coefficient useful for HPLC analysis, 4)
DEDTC is readily available and is
inexpensive, 5) the increased
hydrophobic nature of the derivatized
DEDTC improved extraction efficiency, 6)
a wide variety of DEDTC derivatives are
suitably volatile for GC separation, and 7)
no interference was observed with the
Ames Salmonella assay for mutagenicity.
Table 1. Monomeric Nucleophiles Examined as Tagging Reagents
The examination of chlorinated waste
water samples, concentrates of a
chlorinated drinking water and samples of
chlorinated humic acids demonstrated
the presence of a host of labeled
compounds. The dissimilarity of ion
chromatograms for m/z = 116 (base
fragment for DEDTC) from these different
samples suggests that the number and
proportion of reactive electrophiles can
vary greatly with sample type. Labeling
with DEDTC was shown by this work to
be valuable for qualitative screening of
mixtures and for trace analysis of known
electrophiles by single-ion monitoring.
However, the complete analysis of the
unknown electrophile content of samples
is not possible. While the number and
relative concentration of electrophiles,
—even at-trace concentrations in-a-complex
matrix is readily available from the single-
ion chromatographic data, the absence of
molecular ion and high-mass fragments
for many compounds often prevents their
positive identification.
The difficulty encountered in achieving
sufficient separation of the trace amounts
of the these labeled electrophiles from
the complex baseline to obtain
unambiguous spectra by GC/MS
prompted the development of a
nucleophile with basic functionality so
that the matrix could be simplified by pH
controlled extraction or ion-exchange
chromatography. N-methyl-2-
mercaptoimidazole was chosen as the
derivatizing agent because model
adducts from standard small organic
halides possessed adequate volatility and
simple mass spectral characteristics.
Isolation and analysis of the basic fraction
from the mixture of the imidazole and a
chlorinated waste water sample by
Nucleophile
Reason for Selection
Cyanide
Thtocyanate
Acetate
Mercaptoacetic acid
p-N,N-DimQthylaminoaniline
Diethyldithiocarbamate
Sodium Benzene Sulfinate
Benzoyihydrazide
Phenylhydrazine
Phenylstilfonyihydrazide
Relatively good nucleophile, radio-label is available, only a minimum increase in molecular weight so
products should have a high probability of being volatile.
Relatively good nucleophile, radio-label is available.
Modest nucleophile, represents potential component of buffers.
Sulfhydral is a good nucleophile.
Products should be electrochemically active for easy detection, tertiary amine should allow selective
acid extraction or isolation ion-exchange column.
Excellent nucleophile, products should be volatile and provide characteristic MS fragments.
Good nucleophile, recognized reagent for quinone analysis.
Recognized reagent for carbonyl analysis.
Recognized reagent for carbonyl analysis.
Recognized reagent for carbonyl analysis.
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Table 2. Polymeric Nucleophilic Reagents Tested
Polvmer-Bound Nucleophile
Reason for Selection
Controlled Pore Glass/Thiol
(CPGAThiol) Pierce Chemical Co.
Polystyrene-bound sulfinate
Polystyrene-bound dithiocarboxylate
Polystyrene-bound dithiocarbamate
Commercially available, good nucleophile, structural analogy to
glutathione, cleavage from the polymer is possible by acidic, or
perhaps, enzymatic hydrolysis.
Good nucleophile, known reagent type for quinones', reductive
methods are known for cleavage of the sulfone products, easy to
prepare.
Excellent nucleophile, known facile hydrolysis of the bound thioester
addition products, easy to prepare. •
Excellent nucleophile, enhanced cleavage properties, analogy
possible to monomeric carbamates used in other studies.
GC/MS provided mass spectra which
were not easily interpreted. High
resolution mass spectral analyses were
not attempted, but could perhaps prove
useful in identifying these types of
compounds.
Dimethylaminobenzenethiosulfonate
was synthesized and found to react with
known halides to yield substances that
possessed stable molecular ions.
However, even when the label was
extensively purified, products
representing both the expected
thiosulfonates and the corresponding
sulfones were observed, thereby
complicating the analysis. Despite this,
the thiosulfonate was applied to a
concentrate of a chlorinated drinking
water known to contain Ames positive
mutagens and was shown to reduce the
mutagenic response. Analysis of the
products of this reaction resulted in only
a few spectra which were clearly label-
related.
Three modified polymers were
successfully synthesized and
characterized. These solids were shown
to remove electrophiles from aqueous
samples efficiently. Moreover, one olf
these polymers (the dithiocarboxylate)
was shown to remove the majority of
mutagenic activity from a chlorinated
drinking water sample. The electrophiles;
could not be extracted from the polymers
to any degree by organic solvents or
water. Attempts to cleave the label-
electrophile bonds for analysis proved
inefficient; although the percent recovery
of electrophiles from the polymer was as
high as 20 per cent in isolated cases,
lower values were much more typical.
Conclusions
Diethyldithiocarbamate labeling has
been shown to be a valuable method for
qualitative screening of mixtures and for
trace analysis of known electrophiles by
single-ion monitoring GC/MS. However,
the complete analysis of unknown
electrophile content of samples is not
possible using this method. While the
number and relative concentrations of
electrophiles, even at trace
concentrations in a complex matrix is
readily determined from the single-ion
GC/MS data, the absence of molecular
ions and high-mass fragments for many
of the DEDTC-labeled electrophiles
prevents their positive identification.
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Robert M. Carlson is with the University of Minnesota, Duluth, Minnesota 55812.
Frederick C. Kopfler is the EPA Project Officer (see below).
The complete report, entitled "The Isolation and Identification of Electrophilic
Mutagens Produced During Chlorine Disinfection," (Order No. PB 89-214
1181 AS; Cost: $15.95, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Health Effects Research Laboratory
U.S. Environmental Protection Agency
Research Triangle Park, MC
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Official Business
Penalty for Private Use S300
EPA/600/S1-89/006
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