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Reactions of aqueous chlorine and chlorine dioxide with L- tryptophan

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Reactions of aqueous chlorine and chlorine dioxide with L- tryptophan genotoxicity studies and identification of some genotoxic reaction products
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Owusu-Yaw, Joe D., 1953-
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xii, 246 leaves : ill. ; 29 cm.

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Amino acids ( jstor )
Chlorination ( jstor )
Chlorine ( jstor )
Dioxides ( jstor )
Dosage ( jstor )
Mutagenicity ( jstor )
pH ( jstor )
Potable water ( jstor )
Reaction products ( jstor )
Resins ( jstor )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1989.
Bibliography:
Includes bibliographical references (leaves 216-245).
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Typescript.
General Note:
Vita.
Statement of Responsibility:
by Joe. D. Owusu-Yaw.

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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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REACTIONS OF AQUEOUS CHLORINE AND CHLORINE DIOXIDE WITH L-
TRYPTOPHAN: GENOTOXICITY STUDIES AND IDENTIFICATION OF
SOME GENOTOXIC REACTION PRODUCTS










BY





JOE D. QWUSU-YAW


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1989
















ACKNOWLEDGEMENTS

I wish to express my sincere gratitude to the chairman

of my committee, Dr. Cheng-i Wei, for his guidance, support

and encouragement throughout the course of this study. I

would also like to extend my appreciation to the cochair, Dr.

Willis B. Wheeler, for his support both financially and

otherwise. My very special thanks to Drs. Jesse F. Gregory,

Claude McGowan and Simon S. Yu for their kind assistance and

advice and their willingness to serve on my committee.

I also wish to acknowledge the assistance of Dr. John P.

Toth, Mr. Walter Jones and Mr. Samuel Y. Fernando. I also

wish to acknowledge the financial assistance of Dr. Rod

McDavis, the dean for minority affairs and his group at the

graduate school. Last but not least my special thanks to my

spouse Inza Gibson Owusu-Yaw and my kids Tristian and Jocelyn

who have shared with me the frustrations and joys of academic

work; and to my parents and aunt Agnes who made all this

possible.
















TABLE OF CONTENTS



ACKNOWLEDGEMENTS ................................ .....

LIST OF TABLES........................................

LIST OF FIGURES........................................

ABSTRACT ................................................

CHAPTER

I INTRODUCTION ....................................

Background.......................................
Objectives of the Study..........................

II LITERATURE REVIEW...............................

Historical Perspectives.........................

Chemistry of Aqueous Chlorine....................
Reaction with inorganics....................
Reaction with organics......................
Chlorine disinfection........................

Chemistry of Chlorine Dioxide...................
Reaction with inorganics....................
Reaction with organics......................
Chlorine dioxide disinfection...............

Reactions of Aqueous Chlorine with Amino
Acids, Peptides and proteins................

Reactions of Chlorine Dioxide with Amino
Acids, Peptides and Proteins................

Toxicological Assessment........................
Overview................................. ....
Toxicology of chlorine and chlorine
dioxide.................................
Mutagenesis and carcinogenesis..............


iii


Page

ii

vi

ix

xi



1

1
4

6

6

8
9
13
20

25
27
29
32


35


44

49
50

53
62









III MATERIALS AND METHODS........................... 76

Chlorine Demand-Free Water...................... 76

Generation of Aqueous Chlorine.................. 76

Generation of Aqueous Chlorine Dioxide........... 77

Iodometric Titration............................ 78

Reactions of Aqueous Chlorine or Chlorine
Dioxide with L-Tryptophan................... 79
Preliminary reactions of aqueous
chlorine................................. 79
Reactions of aqueous chlorine and chlorine
dioxide.................................. 79

Chlorine Consumption............................ 81

Concentration Techniques........................ 81
Liquid-liquid extraction.................... 81
Amberlite XAD polymeric resin adsorption.... 84
Rotary evaporation.......................... 87

Fractionation of Reaction Product Extracts...... 89
Thin layer chromatography................... 89

Genotoxicity Assays............................. 91
Ames Salmonella/mammalian microsome assay... 91
Sister chromatid exchange (SCE) assay....... 94
Chromosome preparations..................... 95

Gas Chromatograph/Mass Spectrometer/Data
System...................................... 96

IV RESULTS......................................... 98

Mutagenicity of the Reaction Products of
Aqueous Chlorination of L-Tryptophan........ 98
Rotary evaporation.......................... 98
Liquid-liquid extraction.................... 102

Mutagenicity of the Reaction Products of
Aqueous Chlorine or Chlorine Dioxide
with L-Tryptophan........................... 113
Liquid-liquid extraction.................... 113
Amberlite XAD adsorption .................... 118








Fractionation of the Reaction Products of
Aqueous Chlorine or Chlorine Dioxide and
Tryptophan.................................. 135
Thin layer chromatographic analyses of the
chlorination reaction products........... 135

Induction of Sister Chromatid Exchange by the
TLC Fractions of the Reaction Products of
Aqueous Chlorine or Chlorine Dioxide
with Tryptophan............................. 152

Identification of the Reaction Products.......... 155

V DISCUSSION....................................... 161

Reactions of Aqueous Chlorine and Chlorine
Dioxide with Tryptophan..................... 162

Mutagenicity Evaluation......................... 165
Concentration of the reaction products....... 166
Fractionation of the reaction products....... 182
Comparison of liquid-liquid extraction
and Amberlite XAD 2/8 adsorption........ 185
Comparison of aqueous chlorine and chlorine
dioxide.................................. 188

Induction of Sister Chromatid Exchange by
the TLC Subfractions of the Reaction
Products of Aqueous Chlorine and
Chlorine Dioxide with Tryptophan............. 193

Identification of the Reaction Products.......... 196

Implications of the Mutagenicity to
Carcinogenicity of the Findings.............. 202

VI SUMMARY.......................................... 205

APPENDIX.............................................. 208

REFERENCES............................................ 216

BIOGRAPHICAL SKETCH................................... 246















LIST OF TABLES


Table Page

1 Some halogenated compounds formed in
drinking water as a result of water
chlorination................................... 65

2 Mutagenicity of the aqueous concentrate arising
from the reaction of equimolar concentrations
of aqueous chlorine and tryptophan using
Salmonella typhimurium strain TA98............. 100

3 Mutagenicity of the aqueous concentrate arising
from the reaction of equimolar concentrations
of aqueous chlorine and tryptophan using
Salmonella typhimurium strain TA100............. 101

4 Mutagenicity of the liquid-liquid extracts of
the reaction products of aqueous chlorination
of tryptophan using Salmonella typhimurium
strain TA98.................................... 104

5 Mutagenicity of the liquid-liquid extracts of
the reaction products of aqueous chlorination
of tryptophan using Salmonella typhimurium
strain TA100..................................... 106

6 Mutagenicity of the liquid-liquid extracts of
the reaction products arising from the
chlorination of tryptophan using Salmonella
typhimurium strain TA98........................ 110

7 Mutagenicity of the liquid-liquid extracts of
the reaction products arising from the
chlorination of tryptophan using Salmonella
typhimurium strain TA100....................... 111

8 Mutagenicity of the ethyl ether extracts arising
from the aqueous chlorination of tryptophan
using Salmonella typhimurium strain TA98....... 114

9 Mutagenicity of the ethyl ether extracts arising
from the aqueous chlorination of tryptophan
using Salmonella typhimurium strain TA100....... 115








10 Mutagenicity of the ethyl ether extracts arising
from the reaction of aqueous chlorine dioxide
with tryptophan using Salmonella typhimurium
strain TA98.................................... 116

11 Mutagenicity of the ethyl ether extracts arising
from the reaction of aqueous chlorine dioxide
with tryptophan using Salmonella typhimurium
strain TA100..................................... 119

12 Percent distribution of radioactivity after resin
adsorption in the reaction of aqueous chlorine
with tryptophan................................ 121

13 Recovery of radioactivity after resin adsorption
in the reaction of aqueous chlorine and chlorine
dioxide with tryptophan........................ 122

14 Percent distribution of radioactivity after resin
adsorption in the reactions of aqueous chlorine or
chlorine dioxide with tryptophan............... 123

15 Mutagenicity of the Amberlite XAD eluates arising
from control aqueous chlorine, chlorine dioxide
and tryptophan solutions....................... 125

16 Mutagenicity of the Amberlite XAD eluates arising
from the reaction of aqueous chlorine with
tryptophan using Salmonella typhimurium strain
TA98.......................................... 127

17 Mutagenicity of the Amberlite XAD eluates arising
from the reaction of aqueous chlorine with
tryptophan using Salmonella typhimurium strain
TA100.......................................... 131

18 Mutagenicity of the Amberlite XAD eluates arising
from the reaction of aqueous chlorine dioxide
with tryptophan using Salmonella typhimurium
strain TA98.................................... 133

19 Mutagenicity of the Amberlite XAD eluates arising
from the reaction of aqueous chlorine dioxide
with tryptophan using Salmonella typhimurium
strain TA100..................................... 137

20 Distribution of radioactivity in the TLC
subfractions of the reaction products of
aqueous chlorine or chlorine dioxide with
tryptophan...................................... 142


vii








21 Mutagenicity of the TLC subfractions obtained from
the Amberlite XAD eluates of the reaction products
of aqueous chlorination of tryptophan using
Salmonella typhimurium strain TA98............. 144

22 Mutagenicity of the TLC subfractions obtained from
the Amberlite XAD eluates of the reaction products
of aqueous chlorination of tryptophan using
Salmonella typhimurium strain TA100............ 146

23 Mutagenicity of the TLC fraction of the reaction
products of the aqueous chlorination of
tryptophan re-developed on a second plate...... 149

24 Mutagenicity of the TLC subfractions obtained from
the Amberlite XAD eluates of the reaction products
of chlorine dioxide and tryptophan using
Salmonella typhimurium strain TA98............. 151

25 Mutagenicity of the TLC subfractions obtained from
the Amberlite XAD eluates of the reaction products
of chlorine dioxide and tryptophan using
Salmonella typhimurium strain TA100............ 153

26 Sister chromatid exchange frequencies induced by
the TLC subfractions of the reaction products of
aqueous chlorine and chlorine dioxide with
tryptophan...................................... 154

27 Some physical properties of Amberlite XAD-2 and
XAD-8 resins ................................... 175


viii















LIST OF FIGURES


Figure Page

1 Chlorine substitution reactions with some
phenolic compounds and aromatic organic acids.. 18

2 Chlorine substitution reactions with
pyrimidines and purines........................ 19

3 Detailed reaction mechanism of the haloform
reaction....................................... 21

4 Reactions of chlorine with amino acids leading
to the formation of nitriles and aldehydes..... 39

5 Schematic presentation of sample preparation
for mutagenicity assessment in the preliminary
studies......................................... 83

6 Schematic presentation of sample preparation
for mutagenicity assessment of the reactions
of aqueous chlorine or chlorine dioxide with
tryptophan...................................... 88

7 Schematic presentation of sample for thin layer
chromatography and genotoxicity assessment..... 136

8 Reconstructed ion chromatogram of the reaction
products from the methylene chloride extract... 156

9 Reconstructed ion chromatogram of the thin
layer chromatography subfraction of the
reaction products of chlorine with
tryptophan...................................... 159

10 Reconstructed ion chromatogram of the thin
layer chromatography subfraction of the
reaction products of chlorine dioxide with
tryptophan...................................... 160

11 Comparison of liquid-liquid extraction and
Amberlite XAD 2/8 adsorption................... 187









12 Comparison of aqueous chlorine and chlorine
dioxide......................................... 190

13 Mechanism for the reaction of tryptophan with
aqueous chlorine............................... 197

14 Electron impact mass spectra of 1,1,3-tri-
chloropropanone and 1,1,3,3-tetrachloro-
propanone....................................... 199

15 Proposed scheme for the formation of unsatur-
ated alkanoic acids........................... 200

16 Proposed pathway for the formation of chlorin-
ated acetones and aldehydes from resorcinol.... 201

17 Proposed mechanism for the formation of
quinoline from tryptophan...................... 203















Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of
the Requirements of the Degree of
Doctor of Philosophy

REACTIONS OF AQUEOUS CHLORINE AND CHLORINE DIOXIDE WITH L-
TRYPTOPHAN: GENOTOXICITY STUDIES AND IDENTIFICATION OF
SOME GENOTOXIC REACTION PRODUCTS

By

JOE D. OWUSU-YAW

AUGUST 1989


Chairman: Cheng-i Wei
Cochairman: Willis B. Wheeler
Major Department: Food Science and Human Nutrition

Aqueous chlorine and chlorine dioxide reacted readily

with tryptophan at three different molar ratios (1:1, 3:1 and

7:1 disinfectant:tryptophan) to produce complex reaction

mixtures and a dark precipitate. Evaluation of the mutagenic

activity of the reaction products using the Ames Salmonella/

microsome assay indicated that they were mutagenic at all the

three ratios used. The nonvolatile mutagenic reaction

products were mainly acidic and neutral compounds; they were

direct-acting and caused both frameshift and base-pair

substitution mutations. The reaction products were also

capable of increasing the sister chromatid exchange

frequencies in vitro in Chinese hamster ovary cells.








Amberlite XAD polymeric resin adsorption was superior to

liquid-liquid extraction for concentrating small quantities

of organic mutagens from the model reaction mixtures.

At the lower disinfectant doses (1:1 and 3:1), the

reaction products of chlorine dioxide with tryptophan were

either more potent mutagens or were similar to that of aqueous

chlorine; while at the highest ratio (7:1), aqueous chlorine

reaction products were consistently more mutagenic. Thin

layer chromatographic separation of the reaction products and

detection under ultraviolet light revealed the presence of

several blue and green fluorescent compounds which had not

been previously reported. The use of radiolabeled

14C-tryptophan indicated the most potent mutagens were produced

in very minute quantities and represented less than 1% of the

total starting material. Gas chromatography/mass spectrometry

of the reaction products at the 1:1 molar ratio did not

reveal the presence of any chlorinated compounds. The

compounds identified at this ratio included isatin, oxindole

and indolylacetonitrile. When an excess of chlorine (7:1

molar) was used, several chlorinated compounds were

identified. Chloral, 1,1,3,3-tetrachloropropanone and

1,1,3-trichloropropanone are some of the known mutagens and

chloroform precursors identified from these reactions.


xii















CHAPTER I
INTRODUCTION


Background

Chlorine is used on a worldwide basis as a disinfectant

for potable water and for treatment of sewage effluents. It

is also used as an anti-foulant in electric plants and as a

biocide for the treatment of industrial waters and in food

processing (White, 1972; Morris, 1971). The commercial use

of chlorine has greatly improved the quality of life and

prevented the transmission of water-borne diseases throughout

the world. In the food industry, chlorine is intentionally

added to flour for oxidizing and bleaching purposes, and to

cooling and cleaning waters for milk, fish, meats, poultry and

fruits and vegetables (Smith and Arends, 1984; Foegeding,

1983; Kotula et al., 1974; Ranken et al., 1965; Mercer and

Sommers, 1957). Dilute aqueous chlorine solutions may be used

to decontaminate fruits, eggs and vegetables (Dychdala, 1983).

Chlorine is FDA approved and may also come into contact with

foods unintentionally when it is used as a cleaning and

sterilizing agent for food processing equipment and packaging.

Concern about the use of chlorine as a disinfectant for

potable water arose when it was discovered that








2

trihalomethanes (THM) are produced as undesirable side

products as a result of water chlorination practices (Bellar

et al., 1974; Rook, 1974). These concerns were exacerbated

when the National Cancer Institute confirmed that chloroform,

a major trihalomethane, was carcinogenic in laboratory animals

(NCI, 1976). This led the U.S. Environmental Protection

Agency to make an amendment to the National Interim Primary

Drinking Water Regulations that set a maximum contaminant

level of 0.10 mg/L for total THMs (USEPA, 1983). Since this

revelation, other halogenated and non-halogenated byproducts

have been shown to be produced as a result of water

chlorination practices (Coleman et al., 1984; Christman et

al., 1983; Loper et al., 1978; Rook, 1977). In addition to

chloroform, other halogenated byproducts such as the di- and

trichloroacetonitriles and their brominated analogs which are

produced during water chlorination, have also been shown to

be genotoxic (Bull et al., 1986; Daniel et al., 1986;

Weisburger, 1977).

Free, proteinaceous and bound amino acids as well as

fulvic and humic substances present in potable water have been

implicated and confirmed as precursors of chlorination

reaction products (Bierber and Trehy, 1983; Oliver, 1983;

Lytle and Perdue, 1981; Trehy and Bierber, 1981; Christman,

1980). In fact, extracts of chlorinated drinking water, amino

acids and fulvic and humic substances have been shown to be

genotoxic in different short-term assays (Horth et al., 1987;








3

Tan et al., 1987b; Fielding and Horth, 1986; Bull and

Robinson, 1985; Meier et al., 1983; Sussmuth, 1982; Nazar and

Rapson, 1982; Loper, 1980; Nestmann et al., 1980). The

difficulty encountered in the interpretation of these findings

is that other poorly characterized compounds are also present

which may or may not contribute to the observed genotoxicity.

Epidemiological evidence has also been adduced to link certain

cancers of the colon, bladder and rectum to water chlorination

practices (Cantor et al., 1985; Cragle et al., 1985; Crump,

1983; Williamson, 1981; Cantor and McCage, 1978).

The formation of undesirable toxic side products during

chlorination to produce potable water and the need to reduce

total THM levels in drinking water to below 0.10 mg/L, has led

to a search for alternate disinfectants (USEPA, 1983).

Chlorine dioxide has been proposed as an alternative oxidant

and drinking water disinfectant in America and has received

numerous favorable reviews (Knocke et al., 1987; Lauer et al.,

1986; Lykins and Griese, 1986; Masschelein, 1985; Aieta et

al., 1980). This is because, unlike chlorine, chlorine

dioxide produces very little or no THMs in treated water and

it does not react with ammonia commonly found in water to form

chloramines (Federal Register, 1983; Symons et al., 1981;

White, 1972). Even though chlorine dioxide produces much

fewer THMs, its use is limited by cost (about 3 times that of

chlorine) and the possible health hazards associated with its

biological imapct and long-term use which are not known.










Objectives of the Study

Amino acids are ubiquitous in the environment and have

been shown to act as precursors of mutagenic and/or

carcinogenic byproducts in drinking water. Based on the

reactions of chlorine and/or chlorine dioxide with amino

acids, the following general objectives were proposed for this

study:

1. To investigate the mutagenic potential of the

reaction products of the environmentally signi-

ficant amino acid, tryptophan, with different

molar concentrations of aqueous chlorine or

chlorine dioxide using available concentration

techniques and the Ames Salmonella mutagenicity

assay.

2. To compare liquid-liquid extraction and Amberlite

XAD adsorption for concentrating the reaction

products and also to compare the mutagenic

activities of aqueous chlorine and chlorine dioxide

reaction products.

3. To separate the reaction products using different

chromatographic methods and to re-evaluate the

mutagenicity of the fractions using the Ames

Salmonella/microsome assay. In addition, to

investigate the highly mutagenic subfractions for

genotoxicity using the sister chromatid exchange

assay in cultured Chinese hamster ovary cells.








5

4. To identify the reaction products in the highly

mutagenic fractions and propose mechanisms for their

formation.














CHAPTER II
LITERATURE REVIEW


Historical Perspectives

Chlorine is an member of the halogen family and although

it is one of the most widely distributed elements on earth,

it is never found uncombined in nature. It exists in the

earth's crust in the form of soluble salts of potassium,

sodium, calcium and magnesium (Dychdala, 1983; White, 1972).

Chlorine is believed to have been known to medieval

chemists long before it was discovered in its gaseous state

in 1774 by a Swedish chemist named Steele (Baldwin, 1927).

Steele called the gas dephlogisticated muriatic acid and

observed that it was soluble in water and had permanent

bleaching effects on paper, vegetables and flowers. The gas

was liquefied by compression in 1805 and was found to act on

metals and their oxides. Davy declared the gas to be an

element and named it chlorine, derived from the Greek word

chlorouss" which has been translated to mean green, greenish

yellow or yellowish green, based on its color (Baldwin, 1927).

In 1811 Davy discovered chlorine dioxide gas when he poured

a strong acid on sodium chlorate (White, 1972). He observed

that the gas had an intense color similar to that of chlorine

and subsequently named it euchlorine.








7

Even though the bleaching action of chlorine had been

used for a long time, the disinfecting and deodorizing

properties were not known until the first part of the

nineteenth century. Chlorinated lime was used for

disinfecting and deodorizing hospitals and for sewage

treatment in London in 1854 (Dychdala, 1983). In 1881, Koch

demonstrated the bactericidal action of chlorine as

hypochlorite on pure bacterial cultures under controlled

laboratory conditions and in 1886, the American Public Health

Association issued a favorable report on the use of

hypochlorites as disinfectants. In 1894, Traube established

the first guidelines for hypochlorites in water purification

and disinfection (Dychdala, 1983; Hadfield, 1957). Chloride

of lime was first introduced to north America for water

treatment in the early part of the nineteenth century and

within a few years, over 800 million gallons of water had been

purified by chlorination (Dychdala, 1983; Race, 1918). To

date, chlorine has gained worldwide recognition as the

disinfectant of choice for the treatment of potable water.

The use of chlorine in medicine began in 1915 when Dakin

used dilute solutions of sodium hypochlorite for disinfecting

open and infected wounds during World War I. Dakin's original

formulation contained chlorinated lime, sodium carbonate and

boric acid (Dychdala, 1983). The dairy industry was perhaps

the first food processing industry to utilize the disinfecting

and deodorizing properties of chlorine (Mercer and Sommers,








8

1957). In this respect chlorine was used as a sanitizer for

milk bottles and milk equipment. Use of chlorine then spread

to other food industries and it is used in all branches of

food processing including canning, flour, poultry, fish, meat

and fruits and vegetables industries.



Chemistry of Aqueous Chlorine

In water, chlorine gas undergoes rapid hydrolysis to form

hypochlorous acid according to the following equation (Morris,

1946):

Cl2 (g) + H20 > H* + Cl + HOC1 1.

A small amount of a id H+, is produced; however, it tends to

lower the pH of the solution. Other molecular and ionic

species such as H20C1+, OC1 and Cl2 are produced; however,

HOC1 is by far the most reactive (Morris, 1978). Consistent

with industrial practice, the term chlorine will be used in

this text to refer to aqueous solutions of the active chlorine

compounds, OC1", C12, and HOC1. Molecular chlorine gas will

be referred to as such or as chlorine gas. The active

chlorine compounds, OC1 Cl2 and HOCl coexist in solution and

are difficult to distinguish at the trace level, therefore,

they are grouped together as free residual chlorine (White,

1972).

Hypochlorous acid is classified as a weak acid which

tends to undergo partial dissociation as follows:

HOC1 > H + OC1 2.








9

The dissociation constant for equation 2 ranges from 1.6 to

3.2 x 10-8 for the temperature range 0-250C (Morris, 1966).

The amounts and ratios of the different ionic and molecular

species present at any particular time are dependent upon pH,

temperature and chlorinity or dissolved solids. For example,

at pH 6.0 and a temperature of 150C, the fractions of HOC1, Cl2

and OC1 are 0.975, 3.6 x 10-5 and 0.025, respectively.

However, when the pH is increased to 9.0 under identical

conditions, the fractions become 0.038, 1 x 10-9 and 0.962,

respectively (Morris, 1978).

The above-mentioned aqueous chlorine species have

different reactivities which are related to their redox

potentials as follows (Masschelein, 1979):

C12 + 2e > 2C1 (1.36V) 3.

HOC1 + H+ + 2e" > Cl1 + H20 (1.49V) 4.

OC1 + H20 + 2e -> Cl + 20H- (0.9V) 5.

Of the three species, HOC1 is by far the most reactive in the

pH range of 6-7. Morris (1978) has shown that towards

nitrogenous substrates, HOC1 is 300, 800 and 4 x 104 more

reactive than Ci2, H2OC1 and OC1', respectively, in aqueous

solutions at 150C and neutral pH.

Reaction with inorganics

Due to its reactivity, chlorine or hypochlorous acid

readily oxidizes all organic and inorganic materials in

aqueous solutions until at some breakpointt," when demand is

fully satisfied (Dychdala, 1983). Inorganic substances such








10

as carbon, cyanide, iron, nitrite, sulfite, sulfide and

manganese react with hypochlorous acid via oxidation. Either

the chlorine atom or the oxygen atom of hypochlorous acid may

act as the center of reaction. The chlorine atom is more

electropositive, therefore, electrophilic reactions proceed

by way of the chlorine atom (Morris, 1978).

Chlorine reacts rapidly with inorganic carbon according

to the following expression:

C + 2C12 + 2H20 > 4HC1 + CO2 6.

The above reaction takes place in dechlorination processes

which usually employ granular carbon filters, and the acid

formed consumes alkalinity in treated waters (White, 1972).

Reducing substances. Hydrogen sulfide, characterized by its

obnoxious odors, is a constituent of septic sewage and is

commonly found dissolved in underground waters. During

drinking water chlorination, hydrogen sulfide is completely

oxidized to sulfate according to equation 7 (White, 1972):

H2S + 4C12 + 4H20 > H2SO4 + 8HC1 7.

Nitrites are oxidized readily by free available chlorine to

produce nitrates in a reaction similar to the above (Huburt,

1934):

NO2 + HOC1 -> N03 + H+ + Cl 8.

Chlorine reacts with iron in water supplies to remove

iron from water and to produce a coagulant for both water and

sewage treatment. Similarly, under alkaline conditions,

chlorine reacts with cyanide to form cyanate and with soluble








11

manganese compounds to produce insoluble manganese dioxide

which can be removed by filtration. The cyanide process

represents a detoxication mechanism in water treatment

operations (White, 1972).

Ammonia. The reaction of chlorine with ammonia in dilute

aqueous solutions is of practical importance in water

treatment and has been shown to produce three types of

N-chloro compounds or chloramines. In these reactions, the

electrophilic chlorine atom from HOC1 attaches itself to the

bare electron pair of the nitrogen atom with the concurrent

release of H+ from ammonia and OH- from HOC1 to rapidly form

monochloramine and water as shown in equation 9 (Morris,

1978). The reaction can proceed slowly to form dichloramine

and even more slowly to produce trichloramine or nitrogen

trichloride according to the following equations:

HOCl + NH3 > NH2Cl + H20 9.

HOC1 + NH2C1 > NHC12 + H20 10.

HOC1 + NHC12 > NCl3 + H20 11.

The reactions occur in sequence and, therefore, compete with

each other. The chloramines possess some oxidative power and

are collectively referred to as combined residual chlorine.

Factors affecting the types and ratios of chloramines formed

include pH, temperature, contact time and initial chlorine to

ammonia ratio (White, 1972). For example, equation 9 will

rapidly convert all the free chlorine to monochloramine at pH

7-8 when the ratio of chlorine to ammonia is equimolar, i.e.,








12

5:1 by weight or less. When the molar ratio of chlorine to

ammonia is increased, products from equations 10 and 11 can

also be formed depending on the conditions (White, 1972).

Equations 10 and 11 are related to the breakpointt"

phenomenon used in water treatment for the control of

obnoxious tastes and odors. "Breakpoint" chlorination refers

to the use of excess amounts of chlorine beyond the water

demand, thus resulting in residual free available chlorine

(Griffin, 1946). Breakpoint chlorination leads to increased

germicidal efficiency and disappearance of foul tastes and

odors naturally present in water. At or above the breakpoint,

the initial monochloramine starts a disproportionation

reaction to form dichloramine according to equation 10, which

in turn forms trichloramine or nitrogen trichloride as the

chlorine residuals increase further (White, 1972; Griffin,

1944; Griffin and Chamberlin, 1941).

Other halogens. Apart from chlorine, bromine is perhaps the

second most important halogen with regard to water

chlorination. Bromide, a constituent of natural waters and

seawater, reacts rapidly with chlorine or hypochlorite

according equation 12:

Br + HOC1 > HOBr + Cl 12.

The HOBr is a strong electrophile and reacts much more rapidly

than HOC1 (Morris, 1978). A primary difference between

bromine and chlorine systems is that at neutral pH there are

higher concentrations of molecular bromine, Br2, than








13

molecular chlorine. Also, at any given pH, HOBr ionizes much

more rapidly than HOCl (Jolley and Carpenter, 1983). After

reaction with organic, HOBr is reduced to Br" which is

reoxidized to HOBr by residual aqueous chlorine in the water.

The overall result is enhanced reactivity of aqueous chlorine

in the presence of the bromide ion (Morris, 1978).

Upon chlorination of natural waters containing iodide,

chlorine readily oxidizes the iodide according to the

following equation which forms the basis of the iodometric

method for chlorine determination (Jolley and Carpenter, 1983;

Lister and Rosenblum, 1963).

HOC1 + 21' > 12 + Cl + OH" 13.

Molecular iodine hydrolyses rapidly to form hypoiodous acid

which undergoes dissociation according to equation 14:

HOI > H* + OI 14.

Equation 14 becomes important only at high pH and is not of

practical significance in the chlorination of either natural

or wastewater systems (Jolley and Carpenter, 1983).

Reaction with organics

Reaction of chlorine with organic in drinking water

proceeds via oxidative degradation or chlorine incorporation

through electrophilic substitution or addition reactions. The

major known products of the latter reactions are THMs whose

identification in treated water aroused serious health

concerns in the United States and elsewhere (Bellar, et al.,

1974; Rook, 1974).








14

Oxidation. The predominant type of reaction involving

chlorine and organic compounds in natural and process waters

is oxidation. Carbohydrates and related compounds constitute

a class of organic compounds likely to undergo oxidation

reactions in aqueous solutions (Jolley et al., 1978; Whistler

et al., 1966). Heterocyclic aromatic compounds such as the

purines, pyrimidines and indoles have been shown to undergo

oxidative reactions in dilute aqueous chlorine solutions (Lin

and Carlson, 1984; Hoyano et al., 1973). Indole has been

shown to yield oxindole and isatin upon reaction with chlorine

in dilute aqueous solutions (Lin and Carlson, 1984).

Other classes of compounds known to undergo oxidative

reactions with aqueous chlorine or hypochlorite solutions

include proteins and amino acids, sulfur-containing compounds,

lipids, and polynuclear aromatic hydrocarbons (Trehy et al.,

1986; Lin and Carlson, 1984; Oyler et al., 1983; Dychdala,

1983; Ghanbari et al,, 1983; Burleson et al., 1980).

Addition. Addition of chlorine or halogens to organic

compounds may occur by way of reactive double bonds. This

type of reaction is another example of electrophilic attack

in which the rate determining step is believed to be transfer

of Cl to the double bond to give a chloronium ion (Morris,

1978). Olefins and acetylenes represent a class of compounds

that are vulnerable to attack by chlorine. Their major

reaction with chlorine is by addition in which chlorine atoms

and hydroxyl groups are added to the double bonds (Ghanbari








15

et al., 1983; Carlson and Caple, 1978; Jolley et al., 1978;

Morris, 1978; Pitt et al., 1975; Leopold and Mutton, 1959).

For example, aqueous chlorination of oleic acid has been shown

to produce a mixture of chlorohydrins with water playing a

role in product formation (Carlson and Caple, 1978). Chlorine

addition to double bonds is a slow process and proceeds only

when the double bonds involved are activated (Morris, 1978).

Substitution. The two major types of substitution reactions

of chlorine and chlorine compounds involve formation of either

N- or C-chlorinated compounds.

Formation of N-chlorinated compounds. Nitrogen appears as a

natural component of foods and other substances either as

organic or inorganic nitrogen. Since inorganic nitrogen has

been mentioned earlier, the following discussion will focus

only on organic nitrogen. Sources of organic nitrogen in

natural and process waters include amides, amino acids,

indoles, proteins, nucleic acid bases, and their derivatives

as well as pesticides (Jolley et al., 1978). Organic nitrogen

in water exerts a chlorine demand and leads to the formation

of N-chlorinated compounds (White, 1972; Morris, 1967).

The major reaction mechanisms involved in the formation

of N-chlorinated compounds have been discussed by Morris

(1967). With amines, aqueous chlorine reacts to form N-chloro

compounds by a mechanism similar to its reaction with ammonia.

The electrophilic chlorine atom attaches itself to the bare

electron pair of nitrogen, thus releasing water. Conversely,








16

the reaction may be viewed as one of displacement whereby the

nucleophilic amine displaces OH from HOC1 (Morris, 1978).

Amides also appear to react via the same pattern or via the

haloform reaction but at much slower rates. A simplified

scheme of chlorine substitution with organic N-substituted

organic compounds is shown below (Morris, 1967):

R-NH2 + HOC1 > R-NHC1 + H20 15.

R-CO-NH2 + HOC1 > R-CO-NHC1 + H20 16.

In both cases, the rates of chloramination vary

proportionately with the basic strength or nucleophilicity of

the nitrogenous substrate (Morris, 1978). Proteins are more

complex than amines and amides and chlorination of organic

nitrogen from proteinaceous material is a slow and tedious

process. However, proteins may also undergo substitution with

chlorine to form N-chlorinated proteinaceous byproducts

(Jolley et al., 1978; White, 1972).

Formation of C-chlorinated compounds. Formation of

C-chlorinated compounds via chlorine substitution is perhaps

the most widespread in nature (Jolley et al., 1978; Morris,

1975; Carlson et al., 1975). The major substitution reactions

of chlorine involve substitution into aromatic or heterocyclic

compounds and the haloform reaction. Humic and fulvic

substances abound in nature and represent vegetation under

varying degrees of decay. These complex and otherwise poorly

characterized high molecular weight materials contain aromatic

and heterocyclic moeities which readily undergo chlorination








17

reactions in aqueous chlorine solutions when activated (Jolley

et al., 1978).

Some substitution reactions involving phenolic compounds

and aromatic organic acids are presented in Figure 1. As

before, the mechanism of the reaction involves an

electrophilic attack of HOC1 on the phenoxide ion. In this

case the reaction is facilitated by the presence of a negative

charge on the nucleophilic substrate and only when the ring

is "activated" by substituents such as the oxide ion, does

substitution occur (Morris, 1978). Phenols are more reactive

in dilute aqueous chlorine solutions than other aromatic

compounds possessing different substituent groups (Carlson and

Caple, 1978). Substitution may proceed at the activated ortho

or para positions not fully occupied by chlorine atoms.

Further rupture of the aromatic ring may occur by mechanisms

that are not fully understood (Morris, 1978). Heterocyclic

aromatic compounds such as the nucleic acid bases may also

undergo substitution reactions in which they are either

activated or inactivated toward electrophilic attack compared

to benzene (Morris, 1978). An example of the activation

process involves the chlorination of some purines and

pyrimidines. Aqueous chlorination of the pyrimidines,

cytosine and uracil has been shown to lead to the formation

of their corresponding 5-chloro analogs (Figure 2) and other

poorly characterized byproducts (Jolley et al., 1975; Hoyano

et al., 1973; Patton et al., 1972).














+ HOCI ---


+ HOCI ---


+ HOCI ---






OH
+ HOCI --


+ HOH






+ HOH


COOH


+ HOH
Cl





OH
+ HOH


COOH COOH
+ HOCI + HOH
COOH CI HI-a -COOH








Figure 1. Chlorine substitution reactions with some phenolic
compounds and aromatic organic acids (Adapted from
Jolley et al., 1978).










NH2


+ HOCI --


0 C
II II

HN I + HOCI HN!



0
II
HN N I
,NJ|I + HOCI -.

H H


6H, H
CH3


+ HOH






+ HOH


H HN
H H


+ HOH


3 OH C1 CH3

H N N

CH3


Figure 2. Chlorine substitution reactions with pyrimidines
and purines (Adapted from Jolley et al., 1978).


NH2








20

Similar chlorinated products may be obtained with purines

as shown in Figure 2. With the purines, actual formation of

the chlorinated analogs has not been demonstrated in dilute

aqueous chlorine solutions but rather in non-polar solvents

and in acetic acid (Jolley et al., 1978). Sources of nucleic

acid bases in potable water are plankton, bacteria, and plant

and animal matter. Like the nucleic acid bases, pyrroles are

also thought to be greatly activated to undergo chlorine

substitution reactions (Morris, 1978). Pyridines on the other

hand are thought to be sufficiently deactivated so that

reaction with dilute aqueous chlorine solutions is not

expected to occur in the ring (Morris, 1978).

Finally, formation of C-chlorinated compounds may occur

through the haloform reaction (Figure 3) with humic material

leading to the formation of volatile compounds such as

chloroform (Morris, 1975; Rook, 1974).

Chlorine disinfection

Despite much research done in this area, the actual

mechanism of germicidal action of chlorine has not been fully

elucidated. It is well known that even in trace amounts,

chlorine has fast killing action on bacteria and several

theories have been proposed to explain this killing action.

To date, none of the theories has been fully accepted due to

a lack of substantive evidence (Dychdala, 1983; White, 1972).

The proposal that the germicidal action of chlorine is due to

the liberation of nascent oxygen held for some time until it















0
11 OH
R-C-CH3 so
slow


R-C-CHX

HOX

x2


0e

- R-C=CH2]

HOX rather
r fast
X2A


Oe 0
1 OH- II
R-C=CHX ..=B R-C-CH2X
less
slow


0 0
O OOH
R-C-CHX2 least R-C-CX2 -
least
slow




0
II OH
CHX3 + R-C-OH as H +
Fast at
pH 13


0e

R-C=CX2


HOX even
or faster
X2
0
II
R-C-CX3


Figure 3.


Detailed reaction mechanism of the haloform
reaction. X=Halogen (Adapted from Morris, 1985).








22

was finally dispelled. This is because other strong nascent

oxygen producers such as hydrogen peroxide and potassium

permanganate are less effective bactericides than chlorine

(White, 1972). Also, chlorine still possesses germicidal

activity under conditions which exclude direct oxidation of

bacterial protoplasm (Mercer and Somers, 1957). Other

theories, even though attractive, have not received the full

support of researchers in this area. Baker (1926) proposed

that chlorine reacted with bacteria cell membrane proteins to

produce N-chloro compounds which interfere with bacterial cell

metabolism leading to death. The N-chloro theory was taken

a step further by Rudolph and Levine (1941) who concluded that

the bactericidal activity of chlorine or hypochlorites

proceeds in two stages: 1) the penetration of an active

ingredient into the bacterial cell, and 2) the chemical

reaction of this ingredient with the cellular protoplasm to

form toxic N-chloro compounds which destroy the organism.

Mercer and Somers (1957) stated that bacterial death by

chlorine is a poisoning process in which a form of chlorine

combines chemically with the protoplasm of the bacterial cell

to produce toxic organic complexes.

Other proposed mechanisms of chlorine disinfection were

directed at key enzymes in the target organisms. Green and

Stumpf (1946) and later Knox et al. (1948) observed

experimentally that there was a correlation between the effect

of chlorine on bacterial growth and its effect on the rate of








23

glucose oxidation by certain bacteria. They were able to show

that a bacterial suspension became sterile when the organisms

lost their ability to oxidize glucose. The mechanism of

inhibition was thought to involve irreversible oxidation of

the sulfhydryl groups of essential enzymes like

triosephosphate dehydrogenase. The sulfhydryl oxidation

theory received additional support when chlorine was shown to

oxidize sulfhydryl radicals to sulfonyl compounds and sulfides

to sulfates under conditions that simulate water chlorination

(Ingols et al., 1953; Black and Goodson, 1952; Douglas and

Johnson, 1938). Wyss (1962) postulated the theory of

unbalanced growth in which destruction of part of the enzyme

system throws the bacteria out of metabolic balance leading

to death. Studying the effect of chlorine on bacterial

enzymes, Venkobachar and coworkers (1975) observed that in the

bactericidal concentration of chlorine total dehydrogenase

activity in Eschericia coli was markedly affected and the

inhibition correlated with percent bacteria kill. They also

observed that succinyl dehydrogenase activity in the bacteria

was inactivated probably due to the oxidation of sulfhydryl

groups at lower chlorine doses.

Using radiolabelled chlorine, Friberg (1956) observed

that the first contact oxidation reactions of chlorine with

bacterial cells was responsible for bacterial death and not

through chloramine formation. He later demonstrated that

chlorine, even in minute amounts, reacts with and results in








24

destructive permeability changes in bacterial cell walls

resulting in death (Friberg, 1957). Additional mechanisms of

chlorine disinfection have been reported to include induction

of chromosomal aberrations (Ingols, 1958); disruption of

protein synthesis (Bernade et al., 1967); reaction with

purines and pyrimidines of nucleic acids (Hoyano et al.,

1973); oxidative decarboxylation of amino acids (Pereira et

al., 1973); loss of macromolecules due to inhibition of oxygen

uptake and oxidative decarboxylation (Venkobachar et al.,

1975); production of lesions in DNA (Shih and Lederberg,

1976); inhibition of respiration and active nutrient transport

(Camper and McFeters, 1979); disruption of bacterial spore

coats and increasing spore permeability (Foegeding, 1983).

Chlorine has also been reported to inactivate poliovirus by

reacting with the outer protein coat of the virus (Tenno et

al., 1980) or by damaging the internal RNA (Olivieri et al.,

1980; O'Brien and Newman, 1979).

Of the active chlorine species, hypochlorous acid is by

far the most active disinfectant. Its germicidal efficiency

is related to the ease with which it can penetrate cell walls.

The ease of cell penetration is comparable to that of water

which is structurally similar. Also its relatively low

molecular weight and electrical neutrality allow easy cell

penetration (White, 1972). The germicidal efficiency of free

available chlorine depends on other factors such as pH,

temperature, chlorine concentration, length of contact time,








25

and type and concentration of microorganisms. The pH of the

solution is perhaps the most important of all the above

factors because an increase in pH causes a substantial

decrease in biocidal efficiency and vice versa. This is

because at higher pH, hypochlorous acid is converted to

hypochlorite ion (equation 2) which is an inferior biocide.

The hypochlorite ion obtained from hypochlorous acid

dissociation is a relatively poor disinfectant because of its

inability to diffuse through microbial cell walls. The

inability to traverse microbial cell walls is due to the

negative charge associated with it (White, 1972). Combined

available chlorine as chloramines are also less effective

bactericidal agents than chlorine. Chloramines are slow-

acting and require more than a hundred-fold increase in

contact time and much higher concentrations to achieve the

same bacterial lethality as chlorine (Kabler, 1953;

Butterfield, 1948).



Chemistry of Chlorine Dioxide

Chlorine dioxide is a polar gas that readily dissolves

in but does not react with water to produce aqueous chlorine

dioxide. It is a true dissolved gas because it does not react

with water (Masschelein, 1979). The basic reaction of Davy,

who discovered the gas by pouring a strong acid over sodium

chlorate in 1911, is still used today (White, 1972):

NaC1O3 + 2HC1 > ClO2 + NaCl + H20 + Cl 17.








26

Chlorine dioxide has an intense greenish yellow color with a

distinctive odor similar to chlorine but more irritating and

more toxic (White, 1972). One important physical property of

chlorine dioxide is that it is five times more soluble in

water than chlorine. It is extremely volatile and decays by

photodecomposition, volatilization and decay reactions. In

fact, it can easily be removed from aqueous solutions merely

by aeration. It is very unstable and explosive and as such,

cannot be transported but is usually prepared on site (White,

1972).

Chlorine dioxide does not belong to the family of

"available chlorine" compounds, i.e., those compounds that

hydrolyze to form hypochlorous acid, but due to its oxidizing

power it is referred to as having an available chlorine

content of 263 percent. It has a larger oxidation capacity

than HOC1 because it can accept 2.5 times more electrons than

HOC1 (Ingols and Ridenour, 1948). This can be deduced from

reactions involving iodine liberation from iodide in the

iodometric titration method:

C10 + 51' + 4H -> Cli + 2.5I2 + 2H20 18.

HOC1 + 21' > Cl + I2 + OH 19.

The oxidation potential of chlorine dioxide is less than

that of HOC1 as indicated by its redox potential of 0.95V

compared to that of HOC1 in equation 4. The redox potential

of chlorine dioxide and chlorite are presented below

(Masschelein, 1979):










C10 + e- > C102 (0.95V) 20.

C102 + 2H20 + 4e > Cl + 40H (0.78V) 21.

The oxidizing capacity of chlorine dioxide is not all used in

water treatment because in practice chlorine dioxide is

reduced to chlorite according to equation 20. The chlorite

ion is an effective oxidizing agent and is consumed at a much

slower rate in oxidation-reduction reactions.

Reaction with inorqanics

In the treatment of drinking water, chlorine dioxide is

commonly used for the removal of divalent ferrous iron and

manganese and sometimes sulfide through oxidation reactions

(White, 1972). Much higher temperatures are required for

oxidation of trivalent chromium salts to chromate and will

not be discussed here (Masschelein, 1979).

Oxidation of iron. In water treatment, an effective means of

iron removal is through chlorine dioxide oxidation of iron

from the moderately soluble ferrous (II) to the ferric (III)

state as an insoluble ferric hydroxide precipitate which can

be removed by filtration. The above reaction is presented in

equation 22.

ClO2 + FeO + NaOH + H20 > Fe(OH)3 + NaC102 22.

The pH optimum for the above reaction is above 7.0, and is

preferable in the range between pH 8-9. The reaction is a

slow one, and in most cases, chlorine is the chemical of

choice for this type of reaction. This is because chlorine

is cheaper and equally effective as chlorine dioxide (White,








28

1972). Chlorine dioxide has been shown to oxidize organically

bound iron (Masschelein, 1979).

Oxidation of manganese. Manganese is present in many wells

and surface waters and can cause blackness of water at high

concentrations. Even though it is not known to cause any

health problems, the maximum level permitted in water is 0.05

mg/L (Knocke et al., 1987). Chlorine is used for manganese

removal but the reaction is too slow to be desirable.

Chlorine dioxide reacts more rapidly with manganese compounds

than chlorine. In this reaction, manganese (II) is oxidized

to manganese (IV) as insoluble manganese dioxide according to

the following reaction (White, 1972):

2C102 + MnSO4 + 4NaOH > MnO2 + 2NaC102 +

Na2SO4 + 2H20 23.

The stoichiometric dosage of chlorine dioxide required for

manganese oxidation has been established as 2.45 mg/mg Mn2

assuming the reduction byproduct is chloride. However, under

laboratory conditions, much higher doses of chlorine dioxide

are required. Knocke and coworkers (1987) have recently shown

that at least twice the stoichiometric amount of chlorine

dioxide was required to lower manganese levels to less than

0.05 mg/L. Also, when the total organic carbon content of the

water was high, much more chlorine dioxide was required than

permitted by law to limit manganese content to acceptable

levels.








29

Optimum conditions for the reaction have not been fully

elucidated but it is thought that complete oxidation of

manganese occurs at pH values higher than 7 (White, 1972).

Like iron, chlorine dioxide will oxidize organically bound

manganese. The chlorite formed from chlorine dioxide

reduction can also be used to reduce manganese or iron under

similar conditions (Masschelein, 1979).

Oxidation of sulfides. Hydrogen sulfide is found dissolved

in underground waters and has been shown to react with

chlorine to produce sulfates and elemental sulfur. Chlorine

dioxide oxidation of hydrogen sulfide to sulfate occurs

extensively in the pH range 5-9 (White, 1972). The type of

products formed, either elemental sulfur or sulfate, is

dependent on pH and temperature, with higher temperatures and

pH favoring sulfate formation (Dohnalek and FitzPatrick,

1983). Reactions of chlorine with sulfur has fully been

investigated and characterized but the same does not hold true

for chlorine dioxide. Until more data becomes available on

chlorine dioxide oxidation of sulfides, chlorine remains the

chemical of choice for sulfide removal since it is cheaper and

data is available on the type of byproducts likely to be

encountered.

Reaction with organic

Chlorine dioxide is a selective oxidant which reacts with

organic matter via oxidation to produce both volatile and

non-volatile reaction products. In some cases, however, some








30

chlorinated compounds are encountered. This type of

chlorination reaction is thought to be an indirect one and

probably corresponds to a progressive reduction of the dioxide

passing through the hypochlorous acid stage (Masschelein,

1979). These chlorination reactions involving chlorine

dioxide arise presumably due to the presence of chlorine as

a contaminant in the dioxide used (Masschelein, 1979).

Chlorine dioxide does not react with ammonia to form

chloramines; therefore, chloramine toxicity is avoided with

its use. Unsubstituted aromatics, primary amines and many

other organic compounds are unreactive towards chlorine

dioxide (Raugh, 1979).

Oxidation of phenols and odor control. Phenols are ubiquitous

pollutants in water and the environment and have been used as

model compounds for chlorine reactions and haloform formation

(Aieta and Berg, 1986). Oxidation of phenols and phenolic

compounds by chlorine and chlorine dioxide has been

extensively investigated because phenolic tastes and odors are

produced in natural waters as a result of industrial waste

pollution of raw water. Petroleum and wood processing plants,

algae and decaying vegetation are principal sources of

phenolic compounds. Chlorination of polluted waters leads to

the formation of chlorophenols such as 2-chlorophenol ,

2,4-dichlorophenol, 2,4,6-trichlorophenol and others which

possess distinct offensive tastes and odors (White, 1972).








31

Chlorine dioxide has been shown to be specific for the

control of these phenolic and chlorophenolic tastes and odors

many times faster than free available chlorine. This is

related to the high oxidizing capacity of chlorine dioxide

when compared to chlorine (Masschelein, 1979; White, 1972).

Apart from control of phenolic tastes and odors, chlorine

dioxide has successfully been used against algae odors, fishy

tastes, and earthy-musty taste and odor compounds (Lalezary

et al., 1986; Wright, 1980; Medker et al., 1968).

Reaction of equal parts of chlorine dioxide with phenols

has been reported to yield p-benzoquinone and 2-chloro-

benzoquinone in the concentration range 1-1000 mg phenol/L.

Organic acids, mainly maleate and oxalate are also formed if

the ratio of chlorine dioxide to phenol is increased five-fold

(Wajon et al., 1982; Masschelein, 1979). Extended oxidation

with excess chlorine dioxide leads to the formation of

carboxylic acids and, under moderate conditions, quinones.

When excess phenols are used, chlorophenols are formed due to

the slow release of hypochlorous acid which subsequently

reacts with excess phenol (Wajon et al., 1982). Under water

treatment conditions, the chlorine dioxide to phenol ratio

will be greater than unity and the major products likely to

be encountered are p-benzoquinone and simple organic acids.

Other oxidative reactions. Carbohydrates, proteins and amino

acids and other food components have been shown to undergo

mainly oxidative reactions with chlorine dioxide and are








32

discussed later in this chapter. Environmentally significant

compounds such as polynuclear aromatic hydrocarbons (PAHs) and

their heterocylcic analogs also react with chlorine dioxide

to varying degrees. Lin and Carlson (1984) subjected some

environmental heterocycles to dilute aqueous solutions of

chlorine, chloramine and chlorine dioxide and the latter was

found to produce mainly oxygenated byproducts. For example,

chlorine dioxide oxidation of indole produced oxindole and

isatin.

Chlorination reactions. As mentioned earlier, chlorination

or chlorine incorporation is not an intrinsic reaction of

chlorine dioxide. These reactions are secondary reactions

which are thought to occur through the release of hypochlorous

acid from a primary product or from impurities in the chlorine

dioxide (Masschelein, 1979). In reactions involving chlorine

and chlorine dioxide incorporation into lipids, Ghanbari et

al. (1982b) showed that chlorine incorporation was many orders

of magnitude higher with chlorine than chlorine dioxide.

Chlorine dioxide disinfection

Even though the bactericidal properties of chlorine

dioxide were known at the beginning of this century, the

practical uses of said properties were not fully utilized

until the middle of the century. This is because chlorine

dioxide was first applied to water treatment around the middle

of the century and also increased environmental and water

pollution led to a search for alternate and varied








33

disinfectants (Masschelein, 1979; White, 1972). McCarthy

(1944) first recommended the use of chlorine dioxide for the

disinfection of water of low organic content. This is because

increasing the organic content of water decreased the

germicidal effectiveness of chlorine dioxide. Ridenour and

Ingols (1947) showed that chlorine dioxide was as effective

as chlorine for the control of E. coli and other bacterial

populations. These workers were the first to show that unlike

chlorine, the bactericidal efficiency of chlorine dioxide was

unaffected over a wide pH range (from pH 6-10), which makes

it suitable for the disinfection of high pH waters. On the

other hand, the germicidal effectiveness of chlorine dioxide

was found to decrease at low temperatures (Ridenour and

Ambruster, 1949).

Most of the studies regarding chlorine dioxide

disinfection have focused on a comparison with that of

chlorine and, as a result, very little information is

available on its mode of action. Some of the comparative

studies have shown that chlorine dioxide is a more efficient

virucide and bactericide than chlorine (Alvarez and O'Brien,

1982; Aieta et al., 1980; Longley et al., 1980; Sawyer, 1976).

The proposed mechanisms of action of chlorine dioxide on

microbes are similar to those of chlorine; either through a

reaction with macromolecules or through gross physiological

changes. Like chlorine, there is evidence that chlorine

dioxide acts on dehydrogenase enzymes located in bacterial








34

cytoplasmic membranes but this evidence has not been

substantiated.

Chlorine dioxide is reactive towards several compounds

including phenols, some amino acids and proteins and this

reactivity has been used to explain its bactericidal action.

Fujii and Ukita (1957) demonstrated that chlorine dioxide

oxidation of tryptophan produced some indole derivatives of

tryptophan which may explain the bactericidal action of

chlorine dioxide. On the mode of killing action of chlorine

dioxide on bacteria, Bernade and coworkers (1967) concluded

that chlorine dioxide was sufficiently reactive with several

amino acids to alter their structures and thus, react with

cell walls. They also observed that protein synthesis was

abruptly inhibited by the action of chlorine dioxide. Roller

et al. (1980) have reported that under laboratory conditions,

the level of chlorine dioxide used in treatment plants does

not affect DNA or protein synthesis. Contrary to the above,

Alvarez and O'Brien (1982) in their studies on poliovirus

concluded that chlorine dioxide reacted with viral RNA leading

to virus inactivation.

Noss and others (1983) investigated the reactivity of

chlorine dioxide with nucleic acids, proteins and amino acids.

Chlorine dioxide readily and rapidly reacted with the amino

acids cysteine, tyrosine and tryptophan but not with viral

RNA. The authors postulated that the virucidal activity of

chlorine dioxide was probably due to alteration of viral








35

proteins. Further reports by the same authors suggest that

chlorine dioxide reaction with tyrosine appears to be the main

mechanism of action of F2 virus inactivation by chlorine

dioxide (Olivieri et al., 1985).

Another probable mechanism of action of chlorine dioxide

may be due to its interaction with membrane lipids and fatty

acids leading to permeability changes in membranes. Ghanbari

et al. (1982b) showed that chlorine dioxide was highly

reactive towards free fatty acids leading to the formation of

mainly oxidized byproducts. The authors observed that free

fatty acids were more reactive with chlorine dioxide than

their methyl esters. From the foregoing discussion, it is

possible that chlorine dioxide oxidation of membrane lipids

and proteins leads to microbial death. Even though some of

the reactions have been reported to occur in model systems,

the actual mechanisms that take place in the intact

microorganisms leading to death still need to be proven.



Reactions of Aqueous Chlorine with Amino Acids, Peptides
and Proteins

Proteins are complex high molecular weight constituents

of plant and animal matter made up of carbon, nitrogen,

oxygen, hydrogen and, in some cases, sulfur. Of these

elements, carbon, nitrogen and sulfur exert a chlorine demand

(White, 1972). Proteins and protein material from many

sources contribute greatly to the organic nitrogen content of








36

natural waters and pose a problem during water chlorination

(Qualls and Johnson, 1985). Amino acids are water soluble and

are typically found in natural waters at concentrations in the

range 2-400 gg/L (Le Cloirec and Martin, 1985). During water

treatment, the amount of amino acids can increase

substantially due to hydrolysis-depolymerization of proteins

or peptides, or due to microbial metabolism (Le Cloirec and

Martin, 1985). In the food industry, amino acid

concentrations are likely to be much higher in process

cleaning waters, and superchlorination of process waters is

sometimes used to control microbial contamination (Robinson

et al., 1981).

The reactions of chlorine with amino acids, proteins and

peptides have been studied in detail and reported to produce

both oxidized and chlorinated byproducts under different

treatment conditions (Jolley et al., 1978; Pereira et al.,

1973). The first report in the literature on the chlorination

of amino acids was made by Langheld (1909) who reported that

sodium hypochlorite reacted with alpha-amino acids to form

mono- or dichloroamino acids which subsequently decomposed to

form aldehydes. Langheld (1909) observed that the rate of

monochloramine decomposition was affected by the amino group;

primary amino groups decomposed to give aldehydes or ketones

as well as ammonia while secondary amino groups produced

amines instead of ammonia. Dakin et al. (1916) and Dakin

(1916) confirmed Langheld's study and showed that nitriles








37

were formed as major products when excess chlorine was used.

Dakin and colleagues showed that hydrogen cyanide was produced

from glycine, acetonitrile from alanine, isobutyl cyanide from

leucine and cyanobenzene from phenyl acetate. The general

mechanism proposed by Dakin (1916) involves the initial

formation of a dichloroamino acid which undergoes further

decomposition to form a cyanide. The chlorination of several

amino acids including those studied by Dakin and coworkers has

been further investigated (Isaac and Morris, 1983a; Margerum

et al., 1979). These studies showed that at neutral pH all

the amino acids tested readily reacted with chlorine with the

possible involvement of N-chloro compounds as intermediates.

Rydon and Smith (1952) developed a paper chromatographic

method for determining N-chloroamino acid formation during

chlorination. They showed that several amino acids including

glycine, alanine, leucine, serine, threonine, lysine,

phenylalanine and histidine liberated chlorine from the

corresponding N-chloro derivatives which reacted positively

with a starch-potassium iodide spray. Cysteine, cystine,

methionine and tyrosine failed to release chlorine due

probably to the formation of mainly oxidized products.

The importance of pH in product formation was

investigated by Wright (1936) who observed that aldehydes were

formed from alpha-amino acids on reaction with sodium

hypochlorite at neutral pH while nitriles predominated at

lower pH. From his point of view, acidity favors chlorination








38

and alkalinity favors oxidation. Wright showed that under

acidic conditions glycine was first chlorinated, and the

resulting chloroamino acid was then oxidized by any available

chlorine present; whilst under alkaline conditions,

destruction of available chlorine was mainly due to oxidation.

Jolley and Carpenter (1983) also observed that at neutral pH,

monochlorinated amino acids were formed with aqueous chlorine,

while dichloro derivatives were produced as major products

under acidic conditions. Stanbro and Smith (1979) studied the

kinetics and mechanisms of decomposition of N-chloroalanine

in aqueous solution and showed that the decomposition products

included acetaldehyde, ammonia and carbon dioxide and,

depending on pH, pyruvate. Le Cloirec and Martin (1985) have

provided additional evidence to support the general mechanisms

already proposed. A summary of the general reaction

mechanisms leading to the formation of nitriles and aldehydes

(Figure 4) has been provided by Le Cloirec and Martin (1985).

In the reactions leading to the formation of aldehydes

and nitriles, imine-type compounds are known as essential

intermediates but have not been isolated in the chlorination

of individual amino acids. However, in the case of a peptide,

DL-alanyl-DL-leucine, an imine-type compound has been isolated

and characterized (Pereira et al., 1973). Pereira et al.

(1973) also showed that nitriles are major and aldehydes are

minor products of amino acid chlorination reactions. Tan et

al. (1987a) studied the kinetics of the reactions of aqueous

















COOH
I -HC1 -HC1
R.CH -- > R.CH = NCl > R.C m N
I -C02
NC12 Nitrile


HOC1


COOH COOH
I HOC1 I -CO2 HOC1
R.CH -> R.CH CH = NH
I I -HC1
NH2 NHC1


OH
I -NH2Cl
R.CH -- > RCHO
NHC Aldehyde
NHC1 Aldehyde


Figure 4. Reactions of chlorine with amino acids leading to
the formation of nitriles and aldehydes (Adapted
from Le Cloirec and Martin, 1985)










chlorine and several amino acids in different buffers and

observed that all the amino acids were reactive towards

chlorine over a wide pH range. The type of buffer system was

found markedly to affect the reaction rates. These workers

did not identify any of the reaction products but confirmed

previous reports (Isaac and Morris, 1983a) that the reaction

rates of the sulfur-containing amino acids, cysteine and

methionine, as well as tryptophan were too rapid to be

monitored by the iodometric method of titration. Jancangelo

and Olivieri (1985) have also reported that the sulfur-

containing amino acids and the heterocyclic amino acid,

tryptophan, are the most reactive with monochloramine.

The specific functional groups of the amino acids have

been shown to affect the type of products likely to be formed

during chlorination. In the case of the sulfur amino acid,

cysteine, Pereira and colleagues (1973) have shown that the

major products of chlorination were cystine and cysteic acid

while cystine produced only cysteic acid as the end product.

Amino acids with phenyl or heterocyclic groups readily form

chlorinated analogs if the rings are sufficiently activated.

For example tyrosine and tryptophan have been shown to form

the corresponding mono- and dichloro derivatives upon aqueous

chlorination (Trehy et al., 1986; Burleson et al., 1980;

Pereira et al., 1973). Aqueous chlorination of tyrosine has

been reported to yield mono- and dichlorobenzeneacetonitrile

and mono- and dichlorobenzeneacetaldehyde (Trehy et al., 1986;








41

Burleson et al., 1980; Pereira et al., 1973). Major

chlorination products of phenylalanine were previously

reported to be phenylacetonitrile (95%) and phenylacetaldehyde

(5%) but some chlorinated derivatives have been identified as

well (Horth et al., 1987).

In the case of tryptophan, Kirk and Mitchell (1980)

observed the formation of a dark-colored precipitate when

reacted with different concentrations of aqueous chlorine.

Although the reaction products were not fully identified, the

authors isolated and partially characterized a monochlorinated

product. Using structurally similar indole compounds, they

showed that the intact heterocyclic ring was very important

in color formation. Such color changes have been used

colorimetrically to determine chlorine dioxide concentrations

in water upon reaction with tyrosine (Hodgen and Ingols,

1954). Tryptophan has also been reported to produce oxidized

indole derivatives as well as ether extractable chlorinated

analogs (Owusu-Yaw et al., 1989; Trehy et al., 1986).

Nitriles and aldehydes are also produced as a result of

aqueous chlorination of tryptophan (Owusu-Yaw et al., 1989;

Horth et al., 1987).

The products of the reactions of chlorine with peptides

and proteins have been reported to be mainly N-chloro or

N,N-dichloro derivatives (Pereira et al., 1973, Rydon and

Smith, 1952). Rydon and Smith (1952) showed that peptides

and proteins reacted with chlorine gas to form








42

N-chloropeptides which could be detected visually on paper

chromatograms using a starch-potassium iodide spray.

According to the authors, color was developed from the

liberation of chlorine from the N-chloropeptide bond which

reacted with the starch iodide-indicator. Pereira and

coworkers (1973) investigated the reactions of excess chlorine

with the four dipeptides, glycyl-L-alanine, glycyl-DL-

phenylalanine, DL-alanine-DL-leucine and L-valyl-L-valine and

showed that the corresponding N,N-dichloro-dipeptides were the

major reaction products. From their point of view, the amide

nitrogen of the peptide was not attacked, and the peptide bond

itself was relatively resistant to chlorination. These

authors were the first to show that N-chloroimines were

produced by the dehydrochlorination of the N,N-dichloro-

dipeptide DL-alanyl-DL-leucine with ethanolic sodium hydroxide

at ambient temperature.

Glycylglycine has been used as a model for the formation

of N,N-dichloroglycylglycine and N-chloroglycylglycine (Isaac

and Morris, 1985; Qualls and Johnson, 1985; Margerum et al.,

1979). These studies show that the rate of chlorination of

the dipeptide is faster than that of several amino acids and

ammonia and that the peptide will be preferentially

chlorinated over ammonia or the amino acids glycine, alanine,

serine and glutamate in water. Other peptides such as

phenylalanylglycine and glycylglycylalanine have been reported

to produce stable chloramine residuals (Horth et al., 1989).








43

From all the studies cited, it becomes clear that the fastest

initial reactions of peptides with chlorine occur at the

nucleophilic terminal amino groups or the reactive side groups

of amino acids. For example when flour was chlorinated with

excess chlorine gas, the levels of the amino acids,

methionine, threonine, cysteine and histidine in the proteins

decreased by approximately, 68, 41, 32 and 17%, respectively

(Ewart, 1968).

Tan et al. (1987a) studied the kinetics of the reactions

of aqueous chlorine and three peptides and observed that the

rates of reaction of L-glycyl-L-tryptophan and L-tryptophyl-

glycine were too rapid to be measured iodometrically. The

authors suggested that the rapid reaction rates were due

mainly to the reaction of chlorine with the heterocyclic ring

of tryptophan and not with the amino or carboxyl groups. In

the case of aspartame, even though the component amino acids,

aspartate and phenylalanine, were individually reactive

towards chlorine, the dipeptide was not. The authors did not

provide any conclusive evidence as to whether any of the

reaction products were chloramines. Apart from the formation

of N-chloro compounds, Tsen and Kulp (1971) have postulated

that amino acid side chains of proteins are progressively

cleaved by increasing concentrations of chlorine. They

observed that the amount of water-extractable proteins in

chlorinated flour increased as the chlorine concentration was

increased. Chlorine has also been shown to be readily








44

incorporated into proteins from different sources including

shrimp, meat and poultry (Johnston et al, 1983; Cunningham and

Lawrence, 1977); egg albumin, gelatin, casein, and bovine

serum albumin (Baker, 1947; Wright, 1936 and 1926; Milroy,

1916); and flour proteins (Wei et al., 1984).



Reactions of Chlorine Dioxide with Amino Acids,
Peptides and Proteins

Chlorine dioxide has been proposed as an alternate to

chlorine for disinfection purposes and it is therefore

important to understand the kind of reactions and byproducts

likely to be formed with its use, especially proteinaceous

material. The reactions of chlorine dioxide under water

treatment conditions are mainly oxidative and as such, it has

been used for the control of phenolic tastes and odors in

natural waters (White, 1972). Several reports are available

in the literature on the reactions of chlorine dioxide with

amino acids under different conditions, but as pointed by

Masschelein (1979), the conditions of the reaction are very

important.

In earlier investigations, Schmidt and Braunsdorf (1922)

observed that most amino acids were not reactive towards

chlorine dioxide and that the least reactive were glycine,

leucine, serine, alanine, phenylalanine, glutamate,

asparagine, valine and hydroxyproline. The authors found

cystine to be very reactive. Bernade et al. (1967) further








45

investigated the reaction of chlorine dioxide with several

amino acids and observed that after a contact time of 30

minutes, asparagine, histidine, phenylalanine, arginine,

proline and leucine were unreactive. Contrary to the above

findings, Kennaugh (1957) noted that when a mixture containing

tryptophan, tyrosine, proline, threonine, methionine, valine

and lysine were mixed with diaphanol, a solution of 50% acetic

acid saturated with chlorine dioxide, the aromatic amino acids

disappeared after 18 hours. Also, most of the amino acids

were found to react after 3 days and phenylalanine did not

produce any ninhydrin positive material. Noss et al. (1983)

studied the reactivity of equimolar concentrations (2.5 x

104 M) of twenty-two amino acids and four nucleic acids with

chlorine dioxide at neutral pH and found only seven (cysteine,

cystine, proline, histidine, hydroxyproline, tyrosine and

tryptophan) to be reactive. Additional evidence in support

of these studies was provided by Tan et al. (1987b) who showed

that six of twenty-one amino acids were reactive with chlorine

dioxide. Of the six, the reactions of cysteine, tryptophan

and tyrosine were too rapid to be measured by the iodometric

titration method.

From the above and other studies, it is clear that the

reactivity of chlorine dioxide with amino acids and proteins

depends on pH, temperature and contact time, with higher

temperatures and contact times and lower pH favoring the

reaction. Also, specific functional groups of the amino acids








46

and proteins such as phenyl or sulfhydryl and the relative

molar concentrations of reagent and substrate affect the

reactions. For example, glycine and phenylalanine were inert

toward chlorine dioxide at room temperature when equimolar

concentrations of reagent and substrate were used (Noss et

al., 1983). However, the reactions were found to proceed when

excess molar concentrations of chlorine dioxide were used

(Taymaz et al., 1979).

The sulfur-containing amino acids and those with phenyl

or heterocyclic rings have been reported to be the most

reactive with chlorine dioxide (Masschelein, 1979). Of the

amino acids containing sulfur, cystine has been reported to

produce cysteic acid with a cystine disulfoxide intermediate.

The reaction is said to involve the breakage of the sulfur-

sulfur bond of cystine followed by further oxidation

(Masschelein, 1979). Under similar conditions, methionine is

oxidized to the sulfoxide and then to the sulfone. Other

organic sulfides and mercaptans are also known to undergo

similar oxidative reactions with chlorine dioxide.

Chlorine dioxide oxidation of tyrosine is similar to

those of phenols which are especially susceptible to

oxidation. When pure chlorine dioxide is used, no chlorinated

products are obtained (Masschelein, 1979). With chlorine

dioxide, tyrosine has been reported to produce pink

dopaquinone and red dopachrome. This reaction has been used

as a basis for the colorimetric determination of chlorine








47

dioxide concentrations in water (Hodgen and Ingols, 1954).

When excess chlorine dioxide, 3 moles per mole of tyrosine,

is used for an extended period of time, other unidentified

colored products are formed as well. Reaction of 2 molars

excess of chlorine dioxide with phenylalanine at elevated

temperatures has been reported to produce phenylacetic acid,

phenylacetaldehyde, benzoic acid, benzaldehyde and mandelic

acid (Taymaz et al., 1979). No chlorinated products were

identified from the above reaction. When the chlorine dioxide

concentration was increased to 10 excess molar under similar

conditions, no phenylalanine or above-mentioned products were

detected after 6 days. From the foregoing, it is possible

that further oxidation of the initial reaction products takes

place with excess chlorine dioxide thus giving off carbon

dioxide and causing the disappearance of substrate.

The heterocyclic group of compounds is also very

susceptible to chlorine dioxide oxidation leading to the

formation of complex oxidized products. Chlorine dioxide

oxidation of tryptophan produced indoxyl, isatin, indigo red

as well as unidentified polymerization products (Fujii and

Ukita, 1957). Structurally similar indole derivatives such

as indole itself, 3-methyltryptophan, 3-indolelactic acid and

3-methylindole have also been shown to be readily oxidized by

chlorine dioxide, sometimes leading to color and precipitate

formation (Sen et al., 1989; Lin and Carlson, 1984). Indole

is oxidized to oxindole and isatin while 3-methylindole








48

produces 3-methyloxindole and o-formamidoacetophenone (Lin and

Carlson, 1984).

Unlike amino acids, the reaction of chlorine dioxide with

proteins and peptides has not received much attention in model

systems. A review of the available literature has been

documented by Fukayama et al. (1986). Tan et al. (1987b)

studied the kinetics and mutagenicity of three peptides with

chlorine dioxide at room temperature using equimolar

concentrations of reagent and substrate. They observed that

the reactions of two dipeptides L-tryptophyl-glycine and

L-glycyl-L-tryptophan were too fast to be determined by

iodometric titration. They attributed the very fast reaction

rates to the presence of the tryptophan moieties which have

previously been shown to react rapidly with chlorine dioxide

(Fujii and Ukita, 1957). The third dipeptide, aspartame, was

found to be relatively unreactive since less than 10% of the

initial reagent was lost after 2 hours of reaction. The

authors explained that the inertness of this dipeptide with

chlorine dioxide could be attributed to the component amino

acids, aspartate and phenylalanine, which were individually

unreactive towards chlorine dioxide.

Chlorine gas and chlorine dioxide have traditionally been

used to treat flour to improve baking properties and dough

quality. Meredith et al. (1956) treated wheat flour with high

levels of chlorine dioxide and did not find any chlorinated

derivatives of amino acids and proteins. They, however, found








49

that the levels of the amino acids, cysteine and tryptophan

were decreased by 25 and 8%, respectively. Moran and

coworkers (1953) reported that methionine was oxidized to the

corresponding sulfoxides and sulfones and cysteine to cysteic

acid in flour treated with chlorine dioxide. Ewart (1968)

exposed solutions of wheat proteins to chlorine dioxide and

observed the formation of a pink-yellow color due to the

oxidation of tryptophan and tyrosine. Under similar

conditions there was an increase in chlorine dioxide-induced

solubility of flour proteins. In the treatment of wool,

chlorine dioxide has been reported to cause a dissolution of

up to 40% of the proteinaceous matter leading to losses in

weight. These weight losses have been attributed to the

reactivity of amino acid components of the wool with chlorine

dioxide (Masschelein, 1979). Tyrosine, methionine, glutamate

and tryptophan were the most reactive. The reactivity of

tryptophan with chlorine dioxide was found to be greater than

that of tyrosine and usually led to the formation of a reddish

coloration (Masschelein, 1979).



Toxicological Assessment

Evidence already presented suggests that both chlorine

and chlorine dioxide have the capacity to react with a wide

range of organic compounds under different treatment

conditions. Many organic substances in food and potable water

are capable of reacting with chlorine to produce both








50

halogenated and oxidized products. Chlorine dioxide on the

other hand produces mainly oxidized reaction products. Even

the most gentle chlorination treatments are likely to produce

chlorinated and non-chlorinated organic. Chlorination of

organic compounds has been shown to increase their lipophilic

nature and thereby increase their toxicity and/or their

ability to bioaccumulate (Kopperman et al., 1978). This

section will focus on the toxicology of chlorine and compounds

arising from the use of chlorine and chlorine dioxide

disinfection and their possible health hazards.

Overview

Chlorine has been used as the major disinfectant for

water supplies for many years and has been a major factor in

reducing the transmission and spread of numerous waterborne

diseases and infections. Concerns about the use of chlorine

for water treatment came to light when it was discovered that

THMs are produced as a result of water chlorination practices

(Bellar et al., 1974; Rook, 1974). These concerns gained

additional impetus when the National Cancer Institute reported

that chloroform, a major byproduct of chlorination, is

carcinogenic in two laboratory animals (NCI, 1976). Because

of this discovery, the United States Environmental Protection

Agency amended the National Interim Primary Drinking

Regulation which set up guidelines for the control of THM

concentrations in drinking water. A maximum contaminant level

of 0.10 mg/L was set for total THMs in finished drinking water








51

in the United States (Symons et al., 1981). In order to

comply with these guidelines, several researchers in the area

have devoted their efforts to the search for alternate

disinfectants which will produce very little or no THM in

finished drinking water (Condie, 1986; Lykins and Griese,

1986; Symons et al., 1978).

Chlorine dioxide was selected as the alternate oxidant

and disinfectant for drinking water because in most aspects

it is similar or superior to chlorine and it produced very

little or no THM (Knocke et al., 1987; Aieta and Berg, 1986;

Lykins and Griese, 1986; Lauer et al., 1986; Aieta et al.,

1980). Also, it has been in use for many years in several

European countries without any reported adverse effects

(Symons et al., 1978). According to Symons and coworkers, a

1977 survey showed that about 103 facilities were using or had

used chlorine dioxide in the United States. The suggested use

of chlorine dioxide was welcomed by several researchers and

numerous reviews are available regarding its action,

especially for THM control. One of the main disadvantages of

chlorine dioxide is its additional cost to the consumer which

is estimated at about three times that of chlorine. However,

a report by Lykins and Griese (1986) in Evansville, Indiana,

shows that apart from being suitable for THM control, chlorine

dioxide is also cost effective (about $1.77/resident/year).

On the other hand, it becomes clear that the toxicological

problems and possible health hazards associated with chlorine








52

dioxide and its disinfection byproducts are not immediately

known (Condie, 1986).

Unfortunately, THMs are not the only byproducts of

disinfection which pose possible health hazards in drinking

water. Other chlorinated and non-chlorinated organic

compounds, both volatile and non-volatile, have been reported

to be produced during water chlorination (Loper et al., 1985;

Coleman et al., 1983; Cheh et al., 1980; Christman, 1980).

Organic compounds that have been identified in drinking water

are mostly volatile and represent only about 5-10% of the

total organic compounds in drinking water (NAS, 1977). Of the

250 or more compounds previously identified in drinking water,

about 10% have been classified as being carcinogenic and a

good number remain to be assessed for genotoxicity (Kraybill,

1978). According to the latest survey, more than 1,100

organic compounds have been identified in drinking water, most

of which are at or below the gg/L level (Lucas, 1985).

Another group of compounds, haloacetonitriles (HANs),

has been identified in drinking water which has compounded

the problem (Daniel et al., 1986; Bierber and Trehy, 1983;

Oliver, 1983; Trehy and Bierber, 1981). Proteinaceous

material, especially amino acids, as well as fulvic and humic

substances have been implicated and confirmed precursors of

both THMs and HANs upon reaction with chlorine (Bierber and

Trehy, 1983; Oliver, 1983; Rook, 1980). A third class of

compounds which has received very little attention is the








53

relatively high molecular weight compounds which are not

easily amenable to gas chromatographic analysis. This class

of compounds is mostly non-volatile and several have been

shown to be mutagenic in short-term assays (Cheh et al.,

1980). The difficulty encountered in their isolation and

identification is that they are mostly non-volatile and

difficult to identify using current analytical techniques

(Meier et al., 1983). Some are polymerization products

produced from the condensation of several individual compounds

which makes identification more difficult even with up-to-date

instrumentation. Due to these analytical limitations, the

toxicology and possible health risks posed by this class of

compounds still remain unknown.

Toxicology of chlorine and chlorine dioxide

Chlorine forms hypochlorous acid (HOC1) which dissociates

to form hypochlorite ion (OC1) in solution. Under alkaline

conditions, chlorine dioxide disproportionate to form

chlorite (ClO2") and chlorate (Cl03). Most of the current

literature on the toxicology of chlorine dioxide and chlorine

deals with the undesirable byproducts arising from the

reactions of these disinfectants with organic materials in

water. As a result, very little information is available on

the toxicology of the disinfectants themselves or the

endproducts of their decomposition such as chlorite, and

hypochlorite. This section reviews the available work

concerning the toxicology of chlorine and chlorine dioxide,


1








54

as well as their decomposition byproducts, hypochlorite,

chlorate and chlorite.

Chlorine, hypochlorous acid and hypochlorite

Information dealing with the toxicity of chlorine is

somewhat conflicting. Blabaum and Nichols (1956) reported

that no gross abnormalities occurred in mice fed chlorinated

drinking water for 30 or 50 days. Also, a seven generation

study in rats administered chlorine in their drinking water

did not result in any observable adverse toxic or teratogenic

effects. Other reports on the toxicity of chlorine or

chlorinated water indicate that toxicity, if any, is not due

to chlorine itself but probably due to its reaction with

precursors in the stomach. Chang et al. (1981) have reported

that administration of chlorinated water led to the

development of fatty liver in rats with a better than two-fold

increase in liver triglycerides. The acyl groups of

triacylglycerols and phospholipids were also affected. Such

effects were completely reversible when dosing was

discontinued and the animals recovered fully after 10 days.

The induction of fatty liver may be due to the formation of

chloroform with precursor substances in the body since

chloroform has been shown to give rise to this condition

(Cornish, 1980).

No toxic effects were observed in rats and guinea pigs

fed up to 400 ppm of sodium hypochlorite in milk and water

(Cunningham, 1980). At low dose levels, chlorine was actually








55

shown to act as a broad spectrum antibiotic and stimulated the

growth rates of the rats and guinea pigs. However, at very

high doses (2000 ppm), Cunningham observed significant

increases in liver weights probably due to the formation of

fatty liver described by Chang and coworkers (1981). In

earlier studies, Cunningham and Lawrence (1977) fed flour,

chlorinated with 2,000 and 10,000 ppm chlorine, to rats and

observed decreased growth rates and increased liver and kidney

weights. The authors reported similar effects in rats fed

chlorinated flour lipids and proteins (Cunningham and

Lawrence, 1978).

The tumor initiating and/or promoting activities of

chlorine have also been studied but the results are still

inconclusive. Hayatsu and coworkers (1971) observed that

sodium hypochlorite acts as a cocarcinogen in mice and

increased tumor yields when applied in conjunction with a

sub-threshold dose of 4-nitroquinoline-l-oxide. Contrary to

these observations, Pfeiffer (1978) reported that sodium

hypochlorite decreased the carcinogenic activity of

benzo(a)pyrene in mice.

Chlorine has been reported to induce chromosomal

aberrations in mammalian cells in vitro (Mickey and Holden,

1971), and sodium hypochlorite, which is commonly used in

water disinfection, has been shown to inhibit preferentially

the growth of DNA polymerase-deficient strains of E. coli

(Rosenkranz, 1973). These mutagenic events are due to the








56

reaction of chlorine with the DNA of living matter

(Rosenkranz, 1973). Sodium hypochlorite and not hypochlorous

acid (Thomas et al., 1987) has also been reported to be weakly

mutagenic for Salmonella typhimurium in the Ames Salmonella/

microsome assay (Wlodkowski and Rosenkranz, 1975). It

resulted in a base-pair substitution mutation in Salmonella

presumably due to a reaction with purine and pyrimidine bases

which has previously been demonstrated to occur in vitro

(Hoyano et al., 1973). Meier et al. (1983) have demonstrated

that sodium hypochlorite, and not hypochlorous acid, was

capable of inducing sperm-head abnormalities in mice when fed

various chlorine compounds by gavage. From the evidence

provided, it is not clear whether the mutagenic and/or

carcinogenic effects are due to chlorine itself, or due to the

action of some reaction byproducts. It has been reported that

chloroform, di- and trichloroacetic acid as well as di- and

trichloroacetonitrile were formed in vivo in rats following

oral administration of sodium hypochlorite (Mink et al.,

1983). Some of these byproducts may play a role in the

observed genotoxic effects and will be discussed in the latter

section.

Recent studies by Revis and coworkers (1985) have shown

that chlorine residuals similar to those found in drinking

water increased heart rate, heart fibrous tissue and plasma

cholesterol levels in rabbits and pigeons by interacting with

a diet marginal in calcium. They suggested a relationship








57

between water chlorination and hypothyroidism which they

assumed to be caused by the production of chlorinated

products. In earlier studies Revis et al. (1983) also

observed signs of myocardial hypertrophy and arteriosclerosis

in rabbits and pigeons fed chlorine as sodium hypochlorite in

their drinking water. These changes were reversed when

calcium intakes in the diets were restored to normal.

Chlorine, as chloramine, has received very little

attention in terms of its toxicological potential. It has

been reported to be weakly mutagenic in bacteria (Shih and

Lederberg, 1976) but does not produce sperm-head abnormalities

in mice (Meier et al., 1985). In a very recent study, both

mono- and dichloramines and their derivatives were shown to

be mutagenic in the Ames assay (Thomas et al., 1987). The

authors showed that S. typhimurium strain TA100 was more

sensitive to chloramine mutagenesis and that the lipophilic

dichloramines were more active than the corresponding

monochloramines. Chloramine as the synthetic disinfectant,

chloramine-T, has also been reported to cause sister chromatid

exchanges in cultured Chinese hamster ovary cells in vitro

(Weitberg, 1987). Chloramine has also been evaluated for

teratogenicity in developing rat fetuses but showed little or

no adverse effects at levels up to 100 ppm (Abdel-Rahman et

al., 1982).










Chlorine dioxide, chlorate and chlorite

The toxicology of chlorine dioxide as well its inorganic

byproducts, chlorate (C103) and chlorite (CIO'2), has been

studied extensively because of their observed effects on the

hematopoietic system. All three compounds are usually

evaluated together because both chlorine dioxide and chlorate

can rapidly be converted to chlorite, and therefore, some

aspects of their toxicology are expected to be similar

(Condie, 1986). Like chlorine, considerable disagreement

exists in the literature concerning the toxicity of chlorine

dioxide and its reaction products. Chronic administration of

chlorine dioxide and chlorate to rats in their drinking water

did not produce any observed toxic effects at doses up to 10

mg/L. However, higher mortality rates were observed in rats

administered 100 mg/L for 2 years (Haag, 1949).

Chlorite has been shown to be a potent stressor of blood

and produces methemoglobinemia and hemolytic anemia both in

vivo and in vitro (Hefferman et al., 1979a; 1979b). The dose

required to produce hemolytic anemia is much lower than that

required for methemoglobinemia. It has also been reported to

cause hemolytic anemia, increase osmotic fragility and

glucose-6-phosphate dehydrogenase activity in erythrocytes,

and reduce growth and conception rates in mice (Moore and

Calabrese, 1980a; 1980b; Moore et al., 1980). Further studies

have established that all three compounds produce the same

hemolytic effects in test animals as well as decrease








59

glutathione levels in rats (Abdel-Rahman et al., 1984;

Abdel-Rahman et al., 1979; Couri and Abdel-Rahman, 1979).

Chlorine dioxide induced reduction in glutathione levels

tended to disappear with continued exposure (Couri and

Abdel-Rahman, 1979). The mechanism of the hemolytic activity

of chlorine dioxide and its metabolites has been attributed

to the production of hydrogen peroxide which, like other

strong oxidants, produces this effect (Bull, 1983).

Chlorite and chlorate, but not chlorine dioxide have been

reported to produce hematological effects in nonhuman primates

(Bercz et al., 1982). Chlorine dioxide, on the other hand,

caused a decrease in serum thyroxin levels in monkeys. The

hypothyroid effect was unique to chlorine dioxide since its

endproducts, chlorite and chlorate, did not show this effect

even at higher doses (Bercz et al., 1982). Since chlorine

dioxide is a very reactive oxidant, it is not known whether

the decrease in thyroxin levels is due solely to this

disinfectant or due to some primary oxidation products formed

with precursor molecules in the gastrointestinal tract. Other

studies showed no significant decreases in serum thyroxin

levels in adult rats fed up to 100 mg/L chlorine dioxide

(Condie, 1986). However, administration of similar doses to

pregnant and lactating females resulted in decreased serum

thyroxin concentrations in rat pups (Condie and Bercz, 1985;

Orme et al., 1985).








60

Reproductive and teratogenic studies have also been

performed in laboratory animals. Exposure of female mice to

chlorine dioxide in their drinking water from breeding to

weaning resulted in reduced growth rates and body weights

(Moore et al., 1980), and at much higher concentrations,

increased stillbirths and fetal reabsorptions (Couri et al.,

1982). Contrary to these findings, Suh and coworkers (1983)

did not observe any maternal toxicity or gross changes in

fetal weight, litter size or skeletal anomalies in the

development of rats fed chlorine dioxide, chlorite and

chlorate in their drinking water. Further studies did not

show any measurable adverse reproductive or teratogenic

effects in rats fed sodium chlorite in drinking water (Carlton

and Smith, 1985). Carlton and Smith (1985) showed that

chronic exposure of rats to 100 mg/L sodium chlorite increased

the number of abnormal sperms and to some extent, impaired

sperm motility. This finding is in contrast to that of Meier

and coworkers (1985) who did not detect any sperm-head

abnormalities in mice dosed up to 400 mg/L chlorine dioxide,

chlorate and chlorite.

The effects of chlorine dioxide on the neurobehavioral

development of rats were investigated by Taylor and Pfohl

(1985). They observed that administration of chlorine dioxide

both postnatal and prenatally resulted in behavioral defects

and depressed brain growth. These effects were consistent

with depressed thyroid function. The authors also observed








61

delayed eye opening, decreased body and brain weights and

activities, and delayed locomotor activity. Chlorine dioxide,

and not chlorite, has been implicated as a hyper-

cholesterolemic agent which increased serum cholesterol levels

by interacting with a diet marginal in calcium (Revis et al.,

1985). No definitive conclusions have been made on the

genotoxicity of chlorine dioxide and its metabolites. Suh et

al. (1984) reported that chlorine dioxide depressed DNA

synthesis in the testis and intestinal epithelium of rats

dosed 100 mg/L chlorine dioxide in their drinking water.

Meier et al. (1985) tested the mutagenic activity of chlorine

dioxide, chlorate and chlorite in mice using chromosomal

aberrations, micronucleus and sperm-head abnormalities assays

as endpoints. No mutagenic activity was detected in any of

the disinfectants 5 days after exposure. The tumor initiating

and promoting activities of chlorate and chlorite have also

been investigated. Both chemicals did not demonstrate any

significant carcinogenic activity in Sencar mice (Kurokawa et

al., 1985; 1984).

With regard to human studies, oral administration of

chlorine dioxide, chlorate and chlorite to healthy adult male

volunteers did not produce any detrimental clinical effects

(Lubbers et al., 1985). The authors employed an exposure time

of 12 weeks with the volunteers receiving a total of 5 mg/L

chlorine dioxide or its endproducts. Again, no biochemical

or physiological effects were observed in the volunteers.








62

Caution must be taken in interpreting these findings since a

lack of effect does not necessarily imply safety. It is not

known whether long-term low-dose exposure to any of these

chemicals will induce any severe adverse physiological

effects.

Mutagenesis and Carcinogenesis

Different classes of compounds have been identified in

drinking water, some of which may be produced from

chlorination reactions. A 1976 USEPA survey showed that of

289 compounds identified in drinking water, approximately 38%

were halogenated. Another survey of five major cities in the

United States showed that approximately 50% of the volatile

hydrophobic compounds in drinking water were halogenated

(USEPA, 1975). The number could be considerably higher if

proper analytical methods for detection and quantification

were available. Also, the source and distribution of these

chemicals differ. This section will briefly review the

mutagenic and carcinogenic properties of those compounds that

are formed during water chlorination, such as THMs and HANs,

and those of precursor substances such as amino acids and

humic substances.

Drinking water

The carcinogenic properties of drinking water were first

reported by Marx (1974). Since then numerous studies have

been conducted on the mutagenic and carcinogenic properties

of drinking water in different municipalities in the United








63

States and elsewhere and, in all cases, researchers have shown

that mutagens are produced as a result of water chlorination.

Drinking water in the United States and Canada (Basu et al.,

1985; Nestmann et al., 1983; Cheh et al., 1980; Loper, 1980),

South Africa (Grabow et al., 1980), the Netherlands (Kool et

al., 1985), United Kingdom (Horth et al., 1987; Fielding and

Horth, 1986), Finland (Kronberg et al., 1988), Israel

(Guttman-Bass et al., 1987) and many other countries have been

tested and reported to be mutagenic in several short-term

assays. In most of the studies, replacement of chlorine with

chlorine dioxide leads to a reduction in the observed

mutagenic activities (Guttman-Bass et al., 1987). According

to the findings of Kool et al. (1985), chlorine treatment of

drinking water generally increases the mutagenic activity in

S. typhimurium TA98 and TA100. Also chlorine dioxide is

capable of increasing the mutagenic activity in certain cases

with the overall increase being less than that of chlorine.

The main drawback to these findings is that mutagenicity

is usually detected from a mixture of compounds of different

classes and origin, and even though compounds such as

chloroform, bromoform and tetrachloroethylene are known

carcinogens, their overall contribution to the observed

genotoxicity is not usually known. Being highly volatile,

some of these chemicals may be lost during sample

concentration procedures prior to genotoxicity evaluation.

The third problem is that some of these compounds are poorly








64

characterized non-volatiles which cannot be easily isolated

and identified, therefore, their overall contribution to the

carcinogenic and mutagenic effects remain unknown. None of

the studies in the literature has accounted for 100% of the

mutagenic activity observed in drinking water extracts.

Kronberg et al. (1988) have recently identified a very strong

mutagen,3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone

(MX) and its isomer, (E)-2-chloro-3-(dichloromethyl)-4-

oxobutenoic acid (EMX) in acidified chlorinated extracts of

humic acid and drinking water. According to the authors, MX

accounted for 50-100% of the mutagenic activity of extracts

of chlorinated humic water and for 20-50% of the activity in

drinking water extracts while EMX accounted for only about 2%

of the total mutagenic activities using the Ames Salmonella

assay. MX has also been detected in drinking water from three

locations in the U.S.A. and in the chlorination of humic acid

(Meier et al., 1987). According to the authors, MX appears

to account for a significant proportion of the mutagenic

activity observed in these samples.

Presented in Table 1 is a of some of the compounds

identified in drinking water which includes known or suspected

carcinogens. Volatile compounds have been defined as those

that are purgeable from drinking water at room temperature

with an inert gas such as helium (Tardiff et al., 1978).

These compounds represent 5-10% of the total organic in

drinking water and the remaining 90-95% have not been fully












Table 1. Some halogenated compounds formed in drinking water
as a result of water chlorinationa


Chloroformb

Bromodichloromethaneb

Chlorodibromomethaneb

Bromoformb

Dichloromethane

Bis (2-chloroethyl) etherb

Trichloroethyleneb

Tetrachloroethyleneb

1,1,1-Trichloroethane


Trichloroacetic acid

Trichloroacetaldehyde

Chlorophenols

Alpha-chloroketones

Chlorobenzenes

Chlorinated acetonitrilesb

Chlorinated aromatic acids

Chlorinated purines

Chlorinated pyrimidines


aAdapted from Kraybill (1978)

bKnown or suspected carcinogens










identified, or are unknown (NAS, 1980). Not included are

those high molecular condensation and/or polymerization

products which are not easily amenable to gas chromatographic

analyses even with sample derivatization.

Trihalomethanes and haloacetonitriles

THM were the first class of compounds which aroused

interest in chlorination byproduct formation (Rook, 1974;

Bellar et al., 1974). Most of the purgeable organic are

formed in drinking water at a level of 1 ppb or less.

However, chloroform is usually present in both treated and

untreated water at much higher concentrations of up to 100

ppb on the average (Tardiff et al., 1978; Bellar et al.,

1974). Some of the most frequently encountered chlorinated

compounds in groundwater include trichloroethylene, vinyl

chloride, tetrachloroethylene, 1,1-dichloroethylene, carbon

tetrachloride, 1,1,1-trichloroethane and 1,1-dichlorobenzene

(Cotruvo and Vogt, 1985). Chloroform, the major THM, has been

shown to be carcinogenic and to increase the incidence of

hepatocellular carcinomas and kidney epithelial tumors when

administered to rats and mice (NCI, 1976). However, recent

studies indicate otherwise (Pereira et al., 1985). In 1983,

Pereira showed that THM, including chloroform, possess very

little or no tumor initiating activity and that their main

action in tumorigenesis is promotion. Pereira et al. showed

that chloroform, apart from low level DNA binding, did not

possess any tumor initiating property. In a fairly recent








67
study, Pereira and coworkers (1985) administered 1,800 ppm

chloroform to Swiss mice, pretreated with ethylnitrosourea,

and reported that chloroform inhibited hepatocarcinomas in the

mice. According to the authors, administration of chloroform

in the drinking water of Swiss mice inhibited both spontaneous

and induced hepatocarcinogenesis caused by ethylnitrosourea.

These results are in sharp contrast to the NCI study in which

chloroform was shown to be a hepatocarcinogen. In the NCI

study, chloroform was administered in corn oil and Pereira

and coworkers point out the possibility of a synergistic

interaction between the vehicle and chloroform leading to the

previously observed hepatocarcinogenicity.

Chloroform has been reported to be non-mutagenic in

bacteria assays including Salmonella and E. coli (Simmon et

al., 1977; Uehleke et al., 1976). Other THMs such as

bromoform, bromodichloromethane and dibromochloromethane were

only weakly mutagenic in Salmonella without metabolic

activation (Simmon et al., 1977).

The second major class of volatile organic, HANs, have

received considerable attention since they were discovered

(Oliver, 1983; Bierber and Trehy, 1983). Bierber and Trehy

(1983) reported that HANs easily escape detection because they

undergo gradual hydrolysis to form nonvolatile products.

Dichloroacetonitrile, the major HAN, is usually detected in

drinking water at about 10% that of the average THM

concentration (Oliver, 1983). Haloacetonitriles (HAN) have








68

been reported to pose some possible health problems to humans

because of their potential mutagenic and carcinogenic

properties in test animals (Meier et al., 1983). They have

been detected in the stomach contents of rats dosed with

aqueous chlorine solutions and the possibility exists for the

same effects to be observed in humans (Mink et al., 1983).

Bull and Robinson (1985) tested the mutagenic activity of the

five common HAN, chloroacetonitrile, dichloroacetonitrile,

trichloroacetonitrile, bromochloroacetonitrile, and

dibromoacetonitrile in the Ames Salmonella/microsome assay and

found that dichloroacetonitrile and bromochloroacetonitrile

were direct-acting mutagens which produced base-pair

substitution mutations in Salmonella. All five compounds

induced significant elevations in sister chromatid exchange

(SCE) frequencies in Chinese hamster ovary (CHO) cells. SCE

frequencies increased with increasing chlorine substitution

and was further enhanced when bromine was substituted for

chlorine. Three of the compounds were also capable of

producing tumors in Sencar mice. HAN have been shown to bind

covalently to liver and kidney DNA and cause DNA strand

breaks, which indicates potential carcinogenic ability

(Pereira et al., 1985). Trichloroacetonitrile was found to

be the most potent DNA strand breaker while the bromo-

derivatives were intermediate. Pereira and coworkers have

proposed a scheme for HAN metabolism which involves formation

of phosgene, a highly reactive electrophile, and cyanide.








69

The above findings were further confirmed by Daniel et

al. (1986) who reported that HANs were cytotoxic in human

cultured lymphoblast (CCRF-CEM) cells. They produced 13-60%

cell killing at a dose of 50 gmol and caused major DNA strand

breaks. It should be noted that in all of these studies,

chlorine dioxide has not been mentioned due to the fact that

its use in water treatment does not lead to THM formation.

Further studies are needed to determine the stability of HANs

in aqueous media and the possible health hazards associated

with their long-term low-dose exposure to humans.

Precursor substances

The final part of this review will focus on those

precursor substances which react with chlorine or chlorine

dioxide to form genotoxicants. Proteinaceous material and

humic and fulvic substances are known precursors of

haloacetonitrile and trihalomethane formation in natural

waters upon reaction with chlorine. Unfortunately, these

compounds are not the only ones of concern in drinking water

since other chlorinated and non-chlorinated byproducts are

formed as well. Even though chlorine dioxide produces very

little or no THM in water, it does not remove them from water.

Also, some of the oxidation byproducts of chlorine dioxide

reactions in water may also be genotoxic.

Amino acids, peptides and proteins. The genetic effects of

amino acids after chlorination were first reported by Sussmuth

(1982) who demonstrated that harmless nutrients such as amino








70

acids and yeast and meat extracts could be converted into

carcinogens and mutagens by chlorination. In this experiment,

80 mL of chlorine gas were bubbled through 250 mg of yeast

extract, meat extract or amino acids for 4 minutes and the

reaction products evaluated for genotoxic activity. Due to

the high chlorine dose, Sussmuth did not find the need to

concentrate the reaction products. Methionine was found to

be the most potent precursor of genotoxic activity both in the

Ames Salmonella assay and the rec assay in Bacillus subtilis.

Tyrosine, phenylalanine, cysteine and glycine were also

genotoxic. In the mutagenicity assay, all the above compounds

were direct-acting mutagens which did not require metabolic

activation. None of the reaction products were identified in

this study.

In another study, Horth et al. (1987) evaluated the

mutagenic activity of several amino acids in Salmonella after

reaction with sodium hypochlorite. With the exception of

glycine, mutagenic activity was detected in all the amino

acids treated with chlorine. Methionine, phenylalanine,

tyrosine and a group of heterocyclics were found to be the

most potent precursors of mutagenic activity. Of the

heterocyclics, tryptophan and proline were the most active.

The authors identified several compounds in their mutagenic

extracts. Except for dichloroacetonitrile which is a known

carcinogen, none of the other compounds identified possessed

mutagenic activity when tested separately. It was concluded








71

that the level of dichloroacetonitrile was too low and

unlikely to account for any significant portion of the

mutagenicity. In follow-up studies Horth and coworkers (1989)

tentatively identified several other compounds from

chlorination of tyrosine and phenylalanine. Using authentic

standards, the authors showed that dichloroacetonitrile,

produced from both amino acids, and benzylchloride and

2-phenyl-2,2-dichloroethenal, produced from phenylalanine,

were all mutagenic to S. typhimurium strain TA100.

Dichloroacetonitrile has also been shown to be produced from

the chlorination of aspartic acid, tyrosine and tryptophan

(Trehy et al., 1986). The overall contribution of the

unidentified compounds and those for which no authentic

standards were available still remain unknown.

In similar studies Rapson and coworkers (1985) tested

the mutagenicity of compounds produced by reaction of aqueous

chlorine and chlorine dioxide with tyrosine, at increasing

chlorine to tyrosine ratios. Tyrosine produced mutagenic

reaction products with the mutagenicity increasing with

chlorine dose. Mutagenicity peaked at 4 equivalents of

chlorine per mole of tyrosine. Substitution of chlorine with

chlorine dioxide led to a linear decline in mutagenic activity

from 2,100 revertants over background to 0 with 100% chlorine

dioxide. Similar results were obtained by Tan et al. (1987b)

who showed that replacement of chlorine with chlorine dioxide

on a molar basis decreased the mutagenic activity in








72

chlorinated tryptophan concentrates. However, in the case of

two dipeptides, L-tryptophylglycine and L-glycyl-L-tryptophan,

reaction with chlorine dioxide led to the formation of

mutagens while aqueous chlorine did not (Tan, 1986). In

contrast to the findings of Sussmuth (1982) and Horth et al.

(1987), Tan et al. (1987b) did not detect any mutagenic

activity after reaction of aqueous chlorine and methionine.

Chlorination of purines and pyrimidines as well as

nucleosides and nucleotides did not result in any mutagen

formation (Horth et al., 1987). It is evident that many

difficulties exist in the concentration, fractionation and

evaluation of individual components in drinking water or model

systems. In a majority of tests, a mixture of compounds and

not individual components are used due to analytical

difficulties encountered sample separation. Unless proper

separation and analytical methods are developed, it will be

difficult to pinpoint the individual chemical or group of

chemicals responsible for mutagenic activity in chlorination

reaction mixtures.

Fulvic and humic substances. Fulvic and humic acids are

naturally occurring metabolic endproducts of decaying

vegetation which account for the bulk of organic matter

present in surface waters. Chlorination of water containing

these substances has been reported to result in the formation

of THMs and other non-chlorinated, high molecular weight,

poorly characterized organic (Coleman et al., 1983; Rook,








73

1977), some of which are mutagens and carcinogens (Kronberg

et al., 1988; Meier et al., 1987, 1986 and 1983).

Chlorination of kraft wood pulp, catechol, lignin and

humic and fulvic acids have all been shown to give rise to

mutagen formation in the Ames Salmonella/microsome assay

(Kronberg et al., 1988; Meier et al., 1987; Holmbom et al.,

1984; Kringstad et al., 1983; Meier et al., 1983; Nazar and

Rapson, 1982; Nazar et al., 1981). The pH of the reaction is

important in mutagen formation especially when Amberlite XAD

resins are used to adsorb or concentrate the reaction

mixtures. According to Nazar and Rapson (1982), at elevated

pH of 7 or higher, mutagenic activity decays due to the

cleavage of organically bound chlorine by hydroxide ions.

These findings have been confirmed by Meier et al. (1983) who

observed a decrease in mutagenic activity with increasing pH

due in part to the alkali liability of the compounds.

Mutagenic activity of chlorinated model humic acids is similar

to that of drinking water which implies that these substances

account for a substantial portion of the mutagenic activity

of drinking water (Kronberg et al., 1988; Meier et al., 1987;

Coleman et al., 1984). Also most of the mutagenic activity

is due to non-volatile compounds since most of the volatile

organic are lost during sample concentration (Meier et al.,

1983). Coleman et al. (1984) identified more than 25

compounds from ether extracts of chlorinated humic acids and

confirmed their individual mutagenic activities using








74

authentic standard compounds. Kronberg and colleagues have

also identified and characterized a strong mutagen, MX, from

acidified extracts of chlorinated humic acid using HPLC

fractionation techniques. MX accounted for 50-100% of the

observed mutagenic activity while its isomer, EMX, accounted

for very little of the observed activity. In a separate

study, Meier et al. (1986) identified several compounds from

ether extracts of chlorinated humic acids, some of which are

known mutagens. Analysis of these extracts indicated that

they accounted for only 7% of the total activity in the

original solutions. They also identified MX and EMX which had

previously been identified (Holmbom et al., 1984). Further

studies by Meier et al. (1987) and Hemming et al. (1986)

showed that MX accounted for between 15-34% and 3-19%,

respectively, of the mutagenic activity in XAD concentrates

of drinking water. Both research groups observed that sample

acidification prior to XAD adsorption was essential for the

effective recovery of MX from reaction drinking water or

chlorination reaction mixtures.

In summary, evidence has been provided to show that

genotoxicants are produced during chlorination of water which

contains proteinaceous material and fulvic and humic

substances. A comparison of the mutagenicities of chlorinated

and non-chlorinated surface waters has always shown that

mutagens are present in the chlorinated samples. In any

environmental impact assessment study, those compounds that









75

are not easily identifiable should be taken into account in

order to ensure that they are not being ignored.















CHAPTER III
MATERIALS AND METHODS

Chlorine Demand-free Water

Chlorine demand-free water was prepared following a

modification of the procedure of Ghanbari et al. (1982a).

Distilled water was passed through two successive Barnstead

deionizing units (Barnstead, Division of Sybron Corporation,

Boston, MA) and then through a glass column containing Porapak

Q (Supelco, Inc., Bellefonte, PA). The water was analyzed by

iodometric titration (APHA, 1985) to determine the presence

of any available chlorine. Water used for the preparation of

all the reagents and substrates was prepared this way.



Generation of Aqueous Chlorine

Commercial chlorine is contaminated with a variety of

organochlorine compounds including chloroform, phosgene,

carbon tetrachloride, methylene chloride, etc. (Christman,

1983). To ensure that no such contaminants were present in

the chlorine sample used in this study, a modification of the

methods of Ghanbari et al. (1983) was used for the generation

of aqueous chlorine. Chlorine gas was prepared by dropwise

addition of 3 N HC1 to KMnO4 (Fisher Scientific, Fair Lawn,

NJ) in a closed system under vacuum. The liberated gas was









77

trapped in ice-cold chlorine demand-free water or 0.1 M sodium

phosphate buffer, pH 7 as desired. Under these generation

conditions, the gas was efficiently trapped mainly as

hypochlorous acid. This was detected by a drop in the pH of

the solution from 7.0 to 2.5-3 due to the liberation of

hydrogen ions. The purity and available chlorine

concentrations of the stock solutions were determined by

iodometric titration (APHA, 1985). Aqueous chlorine solutions

were diluted when necessary and used immediately after

preparation.



Generation of Aqueous Chlorine Dioxide

Stock solutions of aqueous chlorine dioxide were prepared

by slow addition of 2 N H2SO4 to 6 g sodium chlorite (Eastman

Kodak Co., Rochester, NY) in 6 mL water in a closed system

(APHA, 1985). The chlorine dioxide gas generated from the

reaction was passed through a sodium chlorite clean-up column

to remove any contaminants such as chlorine. The gas was then

collected as a greenish-yellow solution in ice-cold chlorine

demand-free water or 0.1 M sodium phosphate buffer as desired.

As before, the concentrations of chlorine dioxide solutions

were determined iodometrically. To prevent losses due to

volatilization and/or breakdown, chlorine dioxide solutions

were always prepared fresh, diluted when necessary, and used

immediately after preparation. All chlorine and chlorine








78

dioxide stock solutions were prepared under yellow lights to

prevent photodecomposition.



Iodometric Titration

lodometric titration was used to determine available

chlorine and chlorine dioxide concentrations in all the stock

solutions as well as chlorine residuals remaining after the

reactions. The method involves liberation of iodine from

potassium iodide (KI) by chlorine. The liberated iodine is

then titrated with sodium thiosulfate (Na2S203) to a starch end

point (APHA 1985). Excess KI acidified with acetic acid to

pH 3-4 was mixed with aliquots of the stock chlorine or

chlorine dioxide solutions in glass-stoppered flasks at room

temperature. In the case of chlorine dioxide solutions, the

chlorine dioxide-KI solutions were kept in the dark for about

5 minutes prior to titration. The mixtures were then titrated

under yellow lights with 0.01 N Na2S203 solution prepared by

diluting from standardized stock solutions, until the yellow

color of the liberated iodine was almost clear. A few drops

of a 0.5% starch indicator solution were added and titration

was continued to a colorless end point. A blank titration

using only chlorine demand-free water was also performed to

correct for interference from oxidizing and reducing

substances and to correct for the concentration of iodine

bound starch at the end point (APHA, 1985).








79

The total available chlorine or chlorine dioxide was

calculated as follows:

mg Cl or ClO2 as C12/L (ppm) = (A B) x N x 35450
mL sample


where: A = Volume of Na2S203 used for the sample,

B = Volume of Na2S203 used for the blank,

N = Normality of the Na2S203 used.



Reactions of Aqueous Chlorine or Chlorine Dioxide with
L-Tryptophan

Preliminary reactions of aqueous chlorine

In preliminary experiments, 0.1 M L-tryptophan (Sigma

Chemical Co., St. Louis, MO) solutions were prepared in 0.1

M sodium phosphate buffer at pH 7.0 with slight warming to

ensure that all the amino acid was dissolved. Two molar

ratios of aqueous chlorine to amino acid, 1:1 and 3:1, and a

reaction time of 24 hours was used to ensure that the reaction

was complete. This study used aqueous chlorine alone with pH

adjustment of the reaction mixture after 24 h to obtain

acidic, neutral and basic fractions. Each fraction was

extracted separately first with hexane, then with methylene

chloride and lastly with ethyl ether.

Reactions of aqueous chlorine and chlorine dioxide

In the next series of experiments, three molar ratios of

L-tryptophan to aqueous chlorine or chlorine dioxide, 1:1, 1:3

and 1:7 (M:M), were chosen. Tryptophan solutions were








80

prepared in 0.1 M sodium phosphate buffer, pH 7.0, as before

and aqueous chlorine and chlorine dioxide solutions were

prepared in chlorine demand-free water. For the 1:1 and 1:3

reactions, 14 mM tryptophan solutions (approximately 2.86 g

tryptophan in 1 L sodium phosphate buffer) were reacted with

14 and 42 mM chlorinating agents (total volume 1 L),

respectively. The 1:7 reactions were carried out using a 10

mM amino acid solution (2.04 g tryptophan in 1 L sodium

phosphate buffer) and a 70 mM chlorine or chlorine dioxide

(total volume 1 L). The amino acid concentration used for the

latter reactions were limited by the amount of chlorinating

agent generated by the above procedures. All the reactions

were carried out using equal volumes of reagent and substrate

(2 L final volume), therefore; the final concentrations of

chlorinating agents and amino acid were halved. Reactions

were carried out in the dark in glass jars at ambient

temperature for 24 hours with continuous mixing on a Corning

stirrer. Procedural blanks containing only amino acid,

aqueous chlorine or chlorine dioxide in sodium phosphate

buffer were used as controls.

After reaction, the samples were filtered using a 0.45

gm membrane filter (Gelman Sciences, Inc., Ann Arbor, MI) to

remove any precipitate present. Aliquots of the reaction

mixture were titrated iodometrically to determine if any

available chlorine remained. The samples were then split in

half. The pH of one-half was readjusted to 2.5 with 85%









81

phosphoric acid and saved for resin adsorption, while the

other fraction was kept at neutral pH for liquid-liquid

extraction using ethyl ether.



Chlorine Consumption

Chlorine concentrations before and after chlorination

were measured iodometrically (APHA, 1985). Any excess

chlorine or chlorine dioxide remaining after the 24-hour

reaction was quenched using sodium sulfite (Fisher Scientific,

Fair Lawn, NJ) in order to avoid contamination with elemental

sulfur produced from dechlorination with sodium thiosulfate

(Trehy et al., 1986).



Concentration Techniques

Different techniques, mainly liquid-liquid extraction,

Amberlite XAD adsorption and column chromatography were used

to concentrate the reaction mixtures for mutagenicity and

other assays.

Liquid-liquid extraction

Preliminary studies. In preliminary liquid-liquid extraction

studies, the active ingredients in the aqueous chlorination

reaction mixtures were extracted using organic solvents of

different polarity. Ethyl ether and hexane (Fisher

Scientific) were reagent A.C.S. and methylene chloride (Fisher

Scientific) was HPLC grade. The reaction mixtures containing

aqueous chlorine and tryptophan at neutral pH were extracted









82

three times by mixing vigorously on a stirrer with equal

volumes of hexane, followed by methylene chloride and then

ethyl ether. Each extraction took approximately 2 hours and

represented the neutral fraction. After these extractions,

the aqueous mixture was divided into two portions. The pH of

one fraction was adjusted to 1.2 using concentrated HC1 and

that of the other half was changed to pH 11.7 using 10 N

sodium hydroxide. These fractions were each extracted with

the three organic solvents as before to obtain acidic and

alkaline fractions, respectively, as shown in Fig. 5.

In the preliminary studies, high mutagenic activity was

detected in the ethyl ether extracts of the reaction products.

Therefore, in the latter part of the study when three ratios

of aqueous chlorine and chlorine dioxide were compared, only

ethyl ether was used for the liquid-liquid extractions.

Mixtures containing different molar ratios of aqueous chlorine

or chlorine dioxide with tryptophan were extracted vigorously

with mixing on a stirrer with the aid of a magnetic stirrer

for approximately 4 hours, and then separated with a

separatory funnel. The aqueous phase was discarded and the

ethyl ether extract was dried by rotary evaporation and saved

for further analysis.

Evaluation of product distribution in the ethyl ether

extracts. A solution containing radiolabeled '4C-tryptophan

of known activity was prepared as described below. The

reaction products were extracted with ethyl ether for 2 hours.













0.1 M Aqueous chlorine + 0.1 M tryptophan
in 0.1 M sodium phosphate buffer (pH 7.0)



250C, 24 h


Filter


Precipitate


Aqueous phase



Extract 3x with
-hexane
-methylene chloride
-ethyl ether


Aqueous pha


Adjust to pH 1.2



Extract 3x with
land


Adjust to pH 11.7



hexane, methylene chloride
ethyl ether


\V
-> Concentrate by rotary evaporation
in vacuo



Assay for mutagenic activity


Figure 5. Schematic presentation of sample preparation
for mutagenicity assessment in the preliminary
studies


fl 3 n n =a


y**ac=


__








84

The ethyl ether layer was separated from the aqueous layer and

partially dried to a known volume. The percentage

distribution of radioactivity in the aqueous solution and

ether extracts was measured by dissolving aliquots in 15 mL

Scintiverse II cocktail and counted on a Beckman Instruments

liquid scintillation spectrometer as described below.


Amberlite XAD polymeric resin adsorption

Evaluation of Amberlite XAD procedure for concentrating the

reaction products. Amberlite XAD-2, a nonpolar polystyrene

adsorbent and XAD-8, an acrylic ester of intermediate

polarity, were obtained from Rohm and Haas (Philadelphia, PA).

The resins were prepared prior to use by continuous soxhlet

extraction with acetone for 5 hours followed by further

washing with methanol for an additional 5 hours. The clean

resins were stored separately in methanol until needed. Prior

to use, the resins were slurry packed in methanol in a glass

column, 52 cm by 1.9 cm i.d. to form a bed height of 30 cm.

The resins were then rinsed with 3-5 bed volumes of distilled

water to ensure that all the methanol was removed.

A 7 mM tryptophan solution in 0.1 M sodium phosphate

buffer (pH 7.0) was prepared by dissolving 0.5 g of the amino

acid in 350 mL buffer. Radiolabeled L-tryptophan (side chain-

3-14C) with a specific activity of 53.76 mCi/mmole was obtained

from New England Nuclear, Inc. (Boston, MA). Radiolabeled

tryptophan (0.1 mCi in 0.1 mL phosphate buffer) was mixed with








85

the tryptophan solution and the total radioactivity of the

solution was measured. Equimolar concentrations of either

aqueous chlorine or chlorine dioxide were added to separate

reaction flasks and allowed to react for 4 hours in the dark

at room temperature. The reaction mixtures were filtered

separately using a 0.45 pm membrane filter and the pH and

radioactivity of the solutions were measured. The solutions

were then passed through a glass column (52 cm x 1.9 cm i.d.)

containing either XAD-2 alone or in combination with XAD-8

(1:1 mixture) at a bed height of 30 cm.

Trial experiments were conducted to determine the best

Amberlite XAD concentration technique for the reaction

products. In one experiment, XAD-8 was placed on top of XAD-2

while in another the order was reversed. In another trial,

only XAD-2 was used. Also, the importance of sample pH on the

recovery of the reaction products was evaluated by adjusting

sample pH. The pH of the reaction products in one experiment

was adjusted to 2.5 with 85% phosphoric acid while in another,

it was maintained at 7.0. All the reaction products were

passed through the various resin columns at a flow rate of

approximately 40 mL/minute. The first pass effluent was

poured on the column. The adsorbed substances were eluted

successively with 200 mL each of methanol, ethyl ether and

acetone. The combined organic solvents were removed by rotary

evaporation under vacuum and the residues were diluted to 10

mL with chlorine demand-free water. Radioactivity of the









86

aqueous filtrates and concentrates were measured by counting

0.1 mL aliquots in 15 mL Scintiverse II cocktail (Fisher

Scientific, Fair Lawn, NJ) using a Beckman Instruments

spectrometer Model LS 2800 (Beckman Instruments, Inc.,

Fullerton, CA) with automatic external standardization.

Distribution of radioactivity in the filtrates and eluates

was used to measure the recovery of the reaction products.

Similar sample preparation was used to determine product

distribution in the liquid-liquid extractions.

Due to the high mutagenic activity in the Amberlite

XAD-2/8 acetone eluates of the 7:1 (aqueous chlorine or

chlorine dioxide:tryptophan) ratio, radioactivity distribution

in these eluates were also determined.

Sample concentration for mutagenicity assessment using

Amberlite XAD resins. Based on the results from the

preliminary studies, a resin column with Amberlite XAD-8

placed on top of XAD-2 (1:1 mixture) was used for

concentrating the reaction products. The pH of the aqueous

mixtures containing the reaction products of either chlorine

or chlorine dioxide with tryptophan was adjusted to 2.5 with

85% phosphoric acid and passed through the resins at a flow

rate of approximately 40 mL/minute. This procedure was

repeated using the first-pass effluent. Control samples

containing aqueous chlorine, chlorine dioxide or tryptophan

alone in sodium phosphate buffer were dechlorinated with

sodium sulfite and also concentrated by resin adsorption.









87

After most of the aqueous fraction had passed through the

column and the water level was barely on top of the resin,

acetone was added and allowed to flow freely through the

column until it reached the bottom. Flow was interrupted and

the acetone allowed to equilibrate with the resins for 10-15

minutes. A total of 300 mL of acetone was used to elute most

of the adsorbed substances from the column. Any color or

substances remaining on the column was further eluted

successively with 300 mL methanol followed by 300 mL ethyl

ether. All the eluates were concentrated separately by rotary

evaporation (Fig. 6). The dry samples were weighed, dissolved

in and diluted with spectrophotometric grade dimethyl

sulfoxide (DMSO) and aliquots used for the mutagenicity

studies.

Rotary evaporation

The rotary evaporator used was a Buchi Rotavapor R 110

(Brinkmann Instruments, Inc., Westbury, NY). The aqueous

reaction mixtures containing tryptophan and chlorine as well

as all the organic solvents obtained from the liquid-liquid

extractions and Amberlite XAD adsorptions were concentrated

by rotary evaporation in vacuo at 450C.

Concentrated samples were quantitatively transferred into

preweighed teflon-capped vials and the remaining traces of

organic solvents were removed under a gentle stream of

nitrogen gas. Samples were then weighed, dissolved in and

diluted with spectrophotometric grade DMSO and used for the












Tryptophan solution in sodium phosphate buffer
(pH 7.0)

+

Aqueous chlorine or chlorine dioxide
chlorine to tryptophan ratio (1:1, 3:1 or 7:1)


250C, 24 h


Dechlorinate with sodium sulfite



Filtration



Adjust to pH 2.5 Extract with ether


Amberlite XAD-2/8 adsorption


Elute with-acetone
-methanol
-ether


Concentrate separately by rotary evaporation




Assay for mutagenic activity





Figure 6. Schematic presentation of sample preparation for
mutagenicity assessment in the reactions of
aqueous chlorine or chlorine dioxide with
tryptophan




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AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
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81,9(56,7<


REACTIONS OF AQUEOUS CHLORINE AND CHLORINE DIOXIDE WITH L-
TRYPTOPHAN: GENOTOXICITY STUDIES AND IDENTIFICATION OF
SOME GENOTOXIC REACTION PRODUCTS
BY
JOE D. OWUSU-YAW
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1989

ACKNOWLEDGEMENTS
I wish to express my sincere gratitude to the chairman
of my committee, Dr. Cheng-i Wei, for his guidance, support
and encouragement throughout the course of this study. I
would also like to extend my appreciation to the cochair, Dr.
Willis B. Wheeler, for his support both financially and
otherwise. My very special thanks to Drs. Jesse F. Gregory,
Claude McGowan and Simon S. Yu for their kind assistance and
advice and their willingness to serve on my committee.
I also wish to acknowledge the assistance of Dr. John P.
Toth, Mr. Walter Jones and Mr. Samuel Y. Fernando. I also
wish to acknowledge the financial assistance of Dr. Rod
McDavis, the dean for minority affairs and his group at the
graduate school. Last but not least my special thanks to my
spouse Inza Gibson Owusu-Yaw and my kids Tristian and Jocelyn
who have shared with me the frustrations and joys of academic
work; and to my parents and aunt Agnes who made all this
possible.
11

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS Ü
LIST OF TABLES vi
LIST OF FIGURES ix
ABSTRACT xi
CHAPTER
I INTRODUCTION 1
Background 1
Objectives of the Study 4
II LITERATURE REVIEW 6
Historical Perspectives 6
Chemistry of Aqueous Chlorine 8
Reaction with inorganics 9
Reaction with organics 13
Chlorine disinfection 20
Chemistry of Chlorine Dioxide 25
Reaction with inorganics 27
Reaction with organics 29
Chlorine dioxide disinfection 32
Reactions of Aqueous Chlorine with Amino
Acids, Peptides and proteins 3 5
Reactions of Chlorine Dioxide with Amino
Acids, Peptides and Proteins 44
Toxicological Assessment 49
Overview 50
Toxicology of chlorine and chlorine
dioxide 53
Mutagenesis and carcinogenesis 62
iii

Ill MATERIALS AND METHODS
76
Chlorine Demand-Free Water 7 6
Generation of Aqueous Chlorine 7 6
Generation of Aqueous Chlorine Dioxide 77
Iodometric Titration 78
Reactions of Aqueous Chlorine or Chlorine
Dioxide with L-Tryptophan 7 9
Preliminary reactions of aqueous
chlorine 79
Reactions of aqueous chlorine and chlorine
dioxide 79
Chlorine Consumption 81
Concentration Techniques 81
Liquid-liquid extraction 81
Amberlite XAD polymeric resin adsorption.... 84
Rotary evaporation 8 7
Fractionation of Reaction Product Extracts 89
Thin layer chromatography 89
Genotoxicity Assays 91
Ames Salmonella/mammalian microsome assay... 91
Sister chromatid exchange (SCE) assay 94
Chromosome preparations 95
Gas Chromatograph/Mass Spectrometer/Data
System 96
IV RESULTS 9 8
Mutagenicity of the Reaction Products of
Aqueous Chlorination of L-Tryptophan 98
Rotary evaporation 98
Liquid-liquid extraction 102
Mutagenicity of the Reaction Products of
Aqueous Chlorine or Chlorine Dioxide
with L-Tryptophan 113
Liquid-liquid extraction 113
Amberlite XAD adsorption 118
iv

Fractionation of the Reaction Products of
Aqueous Chlorine or Chlorine Dioxide and
Tryptophan 13 5
Thin layer chromatographic analyses of the
chlorination reaction products 135
Induction of Sister Chromatid Exchange by the
TLC Fractions of the Reaction Products of
Aqueous Chlorine or Chlorine Dioxide
with Tryptophan 152
Identification of the Reaction Products 155
V DISCUSSION 161
Reactions of Aqueous Chlorine and Chlorine
Dioxide with Tryptophan 162
Mutagenicity Evaluation 165
Concentration of the reaction products 166
Fractionation of the reaction products 182
Comparison of liquid-liquid extraction
and Amberlite XAD 2/8 adsorption 185
Comparison of aqueous chlorine and chlorine
dioxide 188
Induction of Sister Chromatid Exchange by
the TLC Subfractions of the Reaction
Products of Aqueous Chlorine and
Chlorine Dioxide with Tryptophan 19 3
Identification of the Reaction Products 196
Implications of the Mutagenicity to
Carcinogenicity of the Findings 202
VI SUMMARY 2 05
APPENDIX 2 08
REFERENCES 216
BIOGRAPHICAL SKETCH 246
v

LIST OF TABLES
Table Page
1 Some halogenated compounds formed in
drinking water as a result of water
chlorination 65
2 Mutagenicity of the aqueous concentrate arising
from the reaction of equimolar concentrations
of aqueous chlorine and tryptophan using
Salmonella tvphimurium strain TA98 100
3Mutagenicity of the aqueous concentrate arising
from the reaction of equimolar concentrations
of aqueous chlorine and tryptophan using
Salmonella tvphimurium strain TA100 101
4 Mutagenicity of the liquid-liquid extracts of
the reaction products of aqueous chlorination
of tryptophan using Salmonella tvphimurium
strain TA98 104
5 Mutagenicity of the liquid-liquid extracts of
the reaction products of aqueous chlorination
of tryptophan using Salmonella tvphimurium
strain TA100 106
6 Mutagenicity of the liquid-liquid extracts of
the reaction products arising from the
chlorination of tryptophan using Salmonella
tvphimurium strain TA98 110
7Mutagenicity of the liquid-liquid extracts of
the reaction products arising from the
chlorination of tryptophan using Salmonella
tvphimurium strain TA100 Ill
8Mutagenicity of the ethyl ether extracts arising
from the aqueous chlorination of tryptophan
using Salmonella tvphimurium strain TA98 114
9Mutagenicity of the ethyl ether extracts arising
from the aqueous chlorination of tryptophan
using Salmonella tvphimurium strain TA100 115
vi

10 Mutagenicity of the ethyl ether extracts arising
from the reaction of aqueous chlorine dioxide
with tryptophan using Salmonella tvphimurium
strain TA98 116
11 Mutagenicity of the ethyl ether extracts arising
from the reaction of aqueous chlorine dioxide
with tryptophan using Salmonella tvphimurium
strain TA100 119
12 Percent distribution of radioactivity after resin
adsorption in the reaction of aqueous chlorine
with tryptophan 121
13 Recovery of radioactivity after resin adsorption
in the reaction of aqueous chlorine and chlorine
dioxide with tryptophan 122
14 Percent distribution of radioactivity after resin
adsorption in the reactions of aqueous chlorine or
chlorine dioxide with tryptophan 12 3
15 Mutagenicity of the Amberlite XAD eluates arising
from control aqueous chlorine, chlorine dioxide
and tryptophan solutions 125
16 Mutagenicity of the Amberlite XAD eluates arising
from the reaction of aqueous chlorine with
tryptophan using Salmonella tvphimurium strain
TA98 127
17 Mutagenicity of the Amberlite XAD eluates arising
from the reaction of aqueous chlorine with
tryptophan using Salmonella tvphimurium strain
TA100 131
18 Mutagenicity of the Amberlite XAD eluates arising
from the reaction of aqueous chlorine dioxide
with tryptophan using Salmonella typhimurium
strain TA98 133
19 Mutagenicity of the Amberlite XAD eluates arising
from the reaction of aqueous chlorine dioxide
with tryptophan using Salmonella tvphimurium
strain TA100 137
20 Distribution of radioactivity in the TLC
subfractions of the reaction products of
aqueous chlorine or chlorine dioxide with
tryptophan 14 2
Vll

21Mutagenicity of the TLC subfractions obtained from
the Amberlite XAD eluates of the reaction products
of aqueous chlorination of tryptophan using
Salmonella typhimurium strain TA98 144
22Mutagenicity of the TLC subtractions obtained from
the Amberlite XAD eluates of the reaction products
of aqueous chlorination of tryptophan using
Salmonella typhimurium strain TA100 146
23 Mutagenicity of the TLC fraction of the reaction
products of the aqueous chlorination of
tryptophan re-developed on a second plate 149
24 Mutagenicity of the TLC subfractions obtained from
the Amberlite XAD eluates of the reaction products
of chlorine dioxide and tryptophan using
Salmonella typhimurium strain TA98 151
25Mutagenicity of the TLC subtractions obtained from
the Amberlite XAD eluates of the reaction products
of chlorine dioxide and tryptophan using
Salmonella typhimurium strain TA100 153
26 Sister chromatid exchange frequencies induced by
the TLC subfractions of the reaction products of
aqueous chlorine and chlorine dioxide with
tryptophan 154
27 Some physical properties of Amberlite XAD-2 and
XAD-8 resins 175
viii

LIST OF FIGURES
Figure Page
1 Chlorine substitution reactions with some
phenolic compounds and aromatic organic acids.. 18
2 Chlorine substitution reactions with
pyrimidines and purines 19
3 Detailed reaction mechanism of the haloform
reaction 21
4 Reactions of chlorine with amino acids leading
to the formation of nitriles and aldehydes 39
5 Schematic presentation of sample preparation
for mutagenicity assessment in the preliminary
studies 83
6 Schematic presentation of sample preparation
for mutagenicity assessment of the reactions
of aqueous chlorine or chlorine dioxide with
tryptophan 88
7 Schematic presentation of sample for thin layer
chromatography and genotoxicity assessment 13 6
8 Reconstructed ion chromatogram of the reaction
products from the methylene chloride extract... 156
9 Reconstructed ion chromatogram of the thin
layer chromatography subfraction of the
reaction products of chlorine with
tryptophan 159
10 Reconstructed ion chromatogram of the thin
layer chromatography subfraction of the
reaction products of chlorine dioxide with
tryptophan 160
11 Comparison of liquid-liquid extraction and
Amberlite XAD 2/8 adsorption 187
IX

12 Comparison of aqueous chlorine and chlorine
dioxide 190
13 Mechanism for the reaction of tryptophan with
aqueous chlorine 197
14 Electron impact mass spectra of 1,1,3-tri-
chloropropanone and 1,1,3,3-tetrachloro-
propanone 199
15 Proposed scheme for the formation of unsatur¬
ated alkanoic acids 200
16 Proposed pathway for the formation of chlorin¬
ated acetones and aldehydes from resorcinol.... 201
17 Proposed mechanism for the formation of
quinoline from tryptophan 203
x

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of
the Requirements of the Degree of
Doctor of Philosophy
REACTIONS OF AQUEOUS CHLORINE AND CHLORINE DIOXIDE WITH L-
TRYPTOPHAN: GENOTOXICITY STUDIES AND IDENTIFICATION OF
SOME GENOTOXIC REACTION PRODUCTS
By
JOE D. OWUSU-YAW
AUGUST 1989
Chairman: Cheng-i Wei
Cochairman: Willis B. Wheeler
Major Department: Food Science and Human Nutrition
Aqueous chlorine and chlorine dioxide reacted readily
with tryptophan at three different molar ratios (1:1, 3:1 and
7:1 disinfectant:tryptophan) to produce complex reaction
mixtures and a dark precipitate. Evaluation of the mutagenic
activity of the reaction products using the Ames Salmonella/
microsome assay indicated that they were mutagenic at all the
three ratios used. The nonvolatile mutagenic reaction
products were mainly acidic and neutral compounds; they were
direct-acting and caused both frameshift and base-pair
substitution mutations. The reaction products were also
capable of increasing the sister chromatid exchange
frequencies in vitro in Chinese hamster ovary cells.
xi

Ainberlite XAD polymeric resin adsorption was superior to
liquid-liquid extraction for concentrating small quantities
of organic mutagens from the model reaction mixtures.
At the lower disinfectant doses (1:1 and 3:1), the
reaction products of chlorine dioxide with tryptophan were
either more potent mutagens or were similar to that of aqueous
chlorine; while at the highest ratio (7:1), aqueous chlorine
reaction products were consistently more mutagenic. Thin
layer chromatographic separation of the reaction products and
detection under ultraviolet light revealed the presence of
several blue and green fluorescent compounds which had not
been previously reported. The use of radiolabeled
KC-tryptophan indicated the most potent mutagens were produced
in very minute quantities and represented less than 1% of the
total starting material. Gas chromatography/mass spectrometry
of the reaction products at the 1:1 molar ratio did not
reveal the presence of any chlorinated compounds. The
compounds identified at this ratio included isatin, oxindole
and indolylacetonitrile. When an excess of chlorine (7:1
molar) was used, several chlorinated compounds were
identified. Chloral, 1,1,3,3-tetrachloropropanone and
1,1,3-trichloropropanone are some of the known mutagens and
chloroform precursors identified from these reactions.
xii

CHAPTER I
INTRODUCTION
Background
Chlorine is used on a worldwide basis as a disinfectant
for potable water and for treatment of sewage effluents. It
is also used as an anti-foulant in electric plants and as a
biocide for the treatment of industrial waters and in food
processing (White, 1972; Morris, 1971). The commercial use
of chlorine has greatly improved the quality of life and
prevented the transmission of water-borne diseases throughout
the world. In the food industry, chlorine is intentionally
added to flour for oxidizing and bleaching purposes, and to
cooling and cleaning waters for milk, fish, meats, poultry and
fruits and vegetables (Smith and Arends, 1984; Foegeding,
1983; Kotula et al., 1974; Ranken et al., 1965; Mercer and
Sommers, 1957). Dilute aqueous chlorine solutions may be used
to decontaminate fruits, eggs and vegetables (Dychdala, 1983).
Chlorine is FDA approved and may also come into contact with
foods unintentionally when it is used as a cleaning and
sterilizing agent for food processing equipment and packaging.
Concern about the use of chlorine as a disinfectant for
potable water arose when it was discovered that
1

2
trihalomethanes (THM) are produced as undesirable side
products as a result of water chlorination practices (Bellar
et al., 1974; Rook, 1974). These concerns were exacerbated
when the National Cancer Institute confirmed that chloroform,
a major trihalomethane, was carcinogenic in laboratory animals
(NCI, 1976). This led the U.S. Environmental Protection
Agency to make an amendment to the National Interim Primary
Drinking Water Regulations that set a maximum contaminant
level of 0.10 mg/L for total THMs (USEPA, 1983). Since this
revelation, other halogenated and non-halogenated byproducts
have been shown to be produced as a result of water
chlorination practices (Coleman et al., 1984; Christman et
al., 1983; Loper et al., 1978; Rook, 1977). In addition to
chloroform, other halogenated byproducts such as the di- and
trichloroacetonitriles and their brominated analogs which are
produced during water chlorination, have also been shown to
be genotoxic (Bull et al., 1986; Daniel et al. , 1986;
Weisburger, 1977) .
Free, proteinaceous and bound amino acids as well as
fulvic and humic substances present in potable water have been
implicated and confirmed as precursors of chlorination
reaction products (Bierber and Trehy, 1983; Oliver, 1983;
Lytle and Perdue, 1981; Trehy and Bierber, 1981; Christman,
1980). In fact, extracts of chlorinated drinking water, amino
acids and fulvic and humic substances have been shown to be
genotoxic in different short-term assays (Horth et al., 1987;

3
Tan et al., 1987b; Fielding and Horth, 1986; Bull and
Robinson, 1985; Meier et al., 1983; Sussmuth, 1982; Nazar and
Rapson, 1982; Loper, 1980; Nestmann et al., 1980). The
difficulty encountered in the interpretation of these findings
is that other poorly characterized compounds are also present
which may or may not contribute to the observed genotoxicity.
Epidemiological evidence has also been adduced to link certain
cancers of the colon, bladder and rectum to water chlorination
practices (Cantor et al., 1985; Cragle et al., 1985; Crump,
1983; Williamson, 1981; Cantor and McCage, 1978).
The formation of undesirable toxic side products during
chlorination to produce potable water and the need to reduce
total THM levels in drinking water to below 0.10 mg/L, has led
to a search for alternate disinfectants (USEPA, 1983).
Chlorine dioxide has been proposed as an alternative oxidant
and drinking water disinfectant in America and has received
numerous favorable reviews (Knocke et al., 1987; Lauer et al.,
1986; Lykins and Griese, 1986; Masschelein, 1985; Aieta et
al., 1980). This is because, unlike chlorine, chlorine
dioxide produces very little or no THMs in treated water and
it does not react with ammonia commonly found in water to form
chloramines (Federal Register, 1983; Symons et al., 1981;
White, 1972). Even though chlorine dioxide produces much
fewer THMs, its use is limited by cost (about 3 times that of
chlorine) and the possible health hazards associated with its
biological imapct and long-term use which are not known.

4
Objectives of the Study
Amino acids are ubiquitous in the environment and have
been shown to act as precursors of mutagenic and/or
carcinogenic byproducts in drinking water. Based on the
reactions of chlorine and/or chlorine dioxide with amino
acids, the following general objectives were proposed for this
study:
1. To investigate the mutagenic potential of the
reaction products of the environmentally signi¬
ficant amino acid, tryptophan, with different
molar concentrations of aqueous chlorine or
chlorine dioxide using available concentration
techniques and the Ames Salmonella mutagenicity
assay.
2. To compare liquid-liquid extraction and Amberlite
XAD adsorption for concentrating the reaction
products and also to compare the mutagenic
activities of aqueous chlorine and chlorine dioxide
reaction products.
3. To separate the reaction products using different
chromatographic methods and to re-evaluate the
mutagenicity of the fractions using the Ames
Salmonella/microsome assay. In addition, to
investigate the highly mutagenic subfractions for
genotoxicity using the sister chromatid exchange
assay in cultured Chinese hamster ovary cells.

5
4. To identify the reaction products in the highly
mutagenic fractions and propose mechanisms for their
formation.

CHAPTER II
LITERATURE REVIEW
Historical Perspectives
Chlorine is an member of the halogen family and although
it is one of the most widely distributed elements on earth,
it is never found uncombined in nature. It exists in the
earth's crust in the form of soluble salts of potassium,
sodium, calcium and magnesium (Dychdala, 1983; White, 1972).
Chlorine is believed to have been known to medieval
chemists long before it was discovered in its gaseous state
in 1774 by a Swedish chemist named Steele (Baldwin, 1927).
Steele called the gas dephlogisticated muriatic acid and
observed that it was soluble in water and had permanent
bleaching effects on paper, vegetables and flowers. The gas
was liquefied by compression in 1805 and was found to act on
metals and their oxides. Davy declared the gas to be an
element and named it chlorine, derived from the Greek word
"chlorous" which has been translated to mean green, greenish
yellow or yellowish green, based on its color (Baldwin, 1927) .
In 1811 Davy discovered chlorine dioxide gas when he poured
a strong acid on sodium chlorate (White, 1972) . He observed
that the gas had an intense color similar to that of chlorine
and subsequently named it euchlorine.
6

7
Even though the bleaching action of chlorine had been
used for a long time, the disinfecting and deodorizing
properties were not known until the first part of the
nineteenth century. Chlorinated lime was used for
disinfecting and deodorizing hospitals and for sewage
treatment in London in 1854 (Dychdala, 1983). In 1881, Koch
demonstrated the bactericidal action of chlorine as
hypochlorite on pure bacterial cultures under controlled
laboratory conditions and in 1886, the American Public Health
Association issued a favorable report on the use of
hypochlorites as disinfectants. In 1894, Traube established
the first guidelines for hypochlorites in water purification
and disinfection (Dychdala, 1983; Hadfield, 1957). Chloride
of lime was first introduced to north America for water
treatment in the early part of the nineteenth century and
within a few years, over 800 million gallons of water had been
purified by chlorination (Dychdala, 1983; Race, 1918). To
date, chlorine has gained worldwide recognition as the
disinfectant of choice for the treatment of potable water.
The use of chlorine in medicine began in 1915 when Dakin
used dilute solutions of sodium hypochlorite for disinfecting
open and infected wounds during World War I. Dakin's original
formulation contained chlorinated lime, sodium carbonate and
boric acid (Dychdala, 1983). The dairy industry was perhaps
the first food processing industry to utilize the disinfecting
and deodorizing properties of chlorine (Mercer and Sommers,

8
1957). In this respect chlorine was used as a sanitizer for
milk bottles and milk equipment. Use of chlorine then spread
to other food industries and it is used in all branches of
food processing including canning, flour, poultry, fish, meat
and fruits and vegetables industries.
Chemistry of Aqueous Chlorine
In water, chlorine gas undergoes rapid hydrolysis to form
hypochlorous acid according to the following equation (Morris,
1946):
Cl2 (g) + H20 > H+ + Cl* + H0C1 1.
A small amount of a id , H+, is produced; however, it tends to
lower the pH of the solution. Other molecular and ionic
species such as H20C1+, 0C1' and Cl2 are produced; however,
H0C1 is by far the most reactive (Morris, 1978). Consistent
with industrial practice, the term chlorine will be used in
this text to refer to aqueous solutions of the active chlorine
compounds, 0C1‘, Cl2, and H0C1. Molecular chlorine gas will
be referred to as such or as chlorine gas. The active
chlorine compounds, 0C1', Cl2 and H0C1 coexist in solution and
are difficult to distinguish at the trace level, therefore,
they are grouped together as free residual chlorine (White,
1972) .
Hypochlorous acid is classified as a weak acid which
tends to undergo partial dissociation as follows:
H0C1
» H + 0C1
2.

9
The dissociation constant for equation 2 ranges from 1.6 to
3.2 x 10s for the temperature range 0-25°C (Morris, 1966).
The amounts and ratios of the different ionic and molecular
species present at any particular time are dependent upon pH,
temperature and chlorinity or dissolved solids. For example,
at pH 6.0 and a temperature of 15°C, the fractions of HOC1, Cl2
and OC1 are 0.975, 3.6 x 10‘5 and 0.025, respectively.
However, when the pH is increased to 9.0 under identical
conditions, the fractions become 0.038, 1 x 10‘9 and 0.962,
respectively (Morris, 1978).
The above-mentioned aqueous chlorine species have
different reactivities which are related to their redox
potentials as follows (Masschelein, 1979):
C]_2 + 2 e
> 2C1
(1.36V)
3 .
HOCl + H+ + 2e' —
>
Cl' + h2o
(1.49V)
4.
OC1' + H20 + 2e -
>
Cl' + 20H
(0.9V)
5.
Of the three species, H0C1 is by far the most reactive in the
pH range of 6-7. Morris (1978) has shown that towards
nitrogenous substrates, H0C1 is 300, 800 and 4 x 104 more
reactive than Cl2, H20C1+, and OC1’, respectively, in aqueous
solutions at 15°C and neutral pH.
Reaction with inorganics
Due to its reactivity, chlorine or hypochlorous acid
readily oxidizes all organic and inorganic materials in
aqueous solutions until at some "breakpoint," when demand is
fully satisfied (Dychdala, 1983) . Inorganic substances such

10
as carbon, cyanide, iron, nitrite, sulfite, sulfide and
manganese react with hypochlorous acid via oxidation. Either
the chlorine atom or the oxygen atom of hypochlorous acid may
act as the center of reaction. The chlorine atom is more
electropositive, therefore, electrophilic reactions proceed
by way of the chlorine atom (Morris, 1978).
Chlorine reacts rapidly with inorganic carbon according
to the following expression:
C + 2C12 + 2H20 > 4HC1 + C02 6.
The above reaction takes place in dechlorination processes
which usually employ granular carbon filters, and the acid
formed consumes alkalinity in treated waters (White, 1972).
Reducing substances. Hydrogen sulfide, characterized by its
obnoxious odors, is a constituent of septic sewage and is
commonly found dissolved in underground waters. During
drinking water chlorination, hydrogen sulfide is completely
oxidized to sulfate according to equation 7 (White, 1972):
H2S + 4C12 + 4H20 > H2S04 + 8HC1 7.
Nitrites are oxidized readily by free available chlorine to
produce nitrates in a reaction similar to the above (Huburt,
1934) :
N02' + HOC1 > N03‘ + H+ + Cl' 8.
Chlorine reacts with iron in water supplies to remove
iron from water and to produce a coagulant for both water and
sewage treatment. Similarly, under alkaline conditions,
chlorine reacts with cyanide to form cyanate and with soluble

11
manganese compounds to produce insoluble manganese dioxide
which can be removed by filtration. The cyanide process
represents a detoxication mechanism in water treatment
operations (White, 1972).
Ammonia. The reaction of chlorine with ammonia in dilute
aqueous solutions is of practical importance in water
treatment and has been shown to produce three types of
N-chloro compounds or chloramines. In these reactions, the
electrophilic chlorine atom from H0C1 attaches itself to the
bare electron pair of the nitrogen atom with the concurrent
release of H+ from ammonia and OH' from H0C1 to rapidly form
monochloramine and water as shown in equation 9 (Morris,
1978) . The reaction can proceed slowly to form dichloramine
and even more slowly to produce trichloramine or nitrogen
trichloride according to the following equations:
H0C1
+
nh3
> nh2ci + h2o
9.
H0C1
+
nh2ci —
> nhci2 + h2o
10.
HOCl
+
nhci2
> nci3 + h2o
11.
The reactions occur in sequence and, therefore, compete with
each other. The chloramines possess some oxidative power and
are collectively referred to as combined residual chlorine.
Factors affecting the types and ratios of chloramines formed
include pH, temperature, contact time and initial chlorine to
ammonia ratio (White, 1972). For example, equation 9 will
rapidly convert all the free chlorine to monochloramine at pH
7-8 when the ratio of chlorine to ammonia is equimolar, i.e.,

12
5:1 by weight or less. When the molar ratio of chlorine to
ammonia is increased, products from equations 10 and 11 can
also be formed depending on the conditions (White, 1972).
Equations 10 and 11 are related to the "breakpoint"
phenomenon used in water treatment for the control of
obnoxious tastes and odors. "Breakpoint" chlorination refers
to the use of excess amounts of chlorine beyond the water
demand, thus resulting in residual free available chlorine
(Griffin, 1946). Breakpoint chlorination leads to increased
germicidal efficiency and disappearance of foul tastes and
odors naturally present in water. At or above the breakpoint,
the initial monochloramine starts a disproportionation
reaction to form dichloramine according to equation 10, which
in turn forms trichloramine or nitrogen trichloride as the
chlorine residuals increase further (White, 1972; Griffin,
1944; Griffin and Chamberlin, 1941).
Other halogens. Apart from chlorine, bromine is perhaps the
second most important halogen with regard to water
chlorination. Bromide, a constituent of natural waters and
seawater, reacts rapidly with chlorine or hypochlorite
according equation 12:
Br' + HOC1 > HOBr + Cl' 12.
The HOBr is a strong electrophile and reacts much more rapidly
than H0C1 (Morris, 1978) . A primary difference between
bromine and chlorine systems is that at neutral pH there are
higher concentrations of molecular bromine, Br2,
than

13
molecular chlorine. Also, at any given pH, HOBr ionizes much
more rapidly than HOC1 (Jolley and Carpenter, 1983). After
reaction with organics, HOBr is reduced to Br which is
reoxidized to HOBr by residual aqueous chlorine in the water.
The overall result is enhanced reactivity of aqueous chlorine
in the presence of the bromide ion (Morris, 1978) .
Upon chlorination of natural waters containing iodide,
chlorine readily oxidizes the iodide according to the
following equation which forms the basis of the iodometric
method for chlorine determination (Jolley and Carpenter, 1983;
Lister and Rosenblum, 1963).
H0C1 + 21 > I2 + Cl' + OH' 13.
Molecular iodine hydrolyses rapidly to form hypoiodous acid
which undergoes dissociation according to equation 14:
HOI > H+ + 01' 14.
Equation 14 becomes important only at high pH and is not of
practical significance in the chlorination of either natural
or wastewater systems (Jolley and Carpenter, 1983).
Reaction with organics
Reaction of chlorine with organics in drinking water
proceeds via oxidative degradation or chlorine incorporation
through electrophilic substitution or addition reactions. The
major known products of the latter reactions are THMs whose
identification in treated water aroused serious health
concerns in the United States and elsewhere (Bellar, et al.,
1974; Rook, 1974).

14
Oxidation. The predominant type of reaction involving
chlorine and organic compounds in natural and process waters
is oxidation. Carbohydrates and related compounds constitute
a class of organic compounds likely to undergo oxidation
reactions in aqueous solutions (Jolley et al., 1978? Whistler
et al., 1966). Heterocyclic aromatic compounds such as the
purines, pyrimidines and indoles have been shown to undergo
oxidative reactions in dilute aqueous chlorine solutions (Lin
and Carlson, 1984; Hoyano et al., 1973). Indole has been
shown to yield oxindole and isatin upon reaction with chlorine
in dilute aqueous solutions (Lin and Carlson, 1984).
Other classes of compounds known to undergo oxidative
reactions with aqueous chlorine or hypochlorite solutions
include proteins and amino acids, sulfur-containing compounds,
lipids, and polynuclear aromatic hydrocarbons (Trehy et al.,
1986; Lin and Carlson, 1984; Oyler et al., 1983; Dychdala,
1983; Ghanbari et al,, 1983; Burleson et al., 1980).
Addition. Addition of chlorine or halogens to organic
compounds may occur by way of reactive double bonds. This
type of reaction is another example of electrophilic attack
in which the rate determining step is believed to be transfer
of Cl+ to the double bond to give a chloronium ion (Morris,
1978). Olefins and acetylenes represent a class of compounds
that are vulnerable to attack by chlorine. Their major
reaction with chlorine is by addition in which chlorine atoms
and hydroxyl groups are added to the double bonds (Ghanbari

15
et al., 1983; Carlson and Caple, 1978; Jolley et al., 1978;
Morris, 1978; Pitt et al., 1975; Leopold and Mutton, 1959).
For example, aqueous chlorination of oleic acid has been shown
to produce a mixture of chlorohydrins with water playing a
role in product formation (Carlson and Caple, 1978). Chlorine
addition to double bonds is a slow process and proceeds only
when the double bonds involved are activated (Morris, 1978).
Substitution. The two major types of substitution reactions
of chlorine and chlorine compounds involve formation of either
N- or C-chlorinated compounds.
Formation of N-chlorinated compounds. Nitrogen appears as a
natural component of foods and other substances either as
organic or inorganic nitrogen. Since inorganic nitrogen has
been mentioned earlier, the following discussion will focus
only on organic nitrogen. Sources of organic nitrogen in
natural and process waters include amides, amino acids,
indoles, proteins, nucleic acid bases, and their derivatives
as well as pesticides (Jolley et al. , 1978) . Organic nitrogen
in water exerts a chlorine demand and leads to the formation
of N-chlorinated compounds (White, 1972; Morris, 1967).
The major reaction mechanisms involved in the formation
of N-chlorinated compounds have been discussed by Morris
(1967). With amines, aqueous chlorine reacts to form N-chloro
compounds by a mechanism similar to its reaction with ammonia.
The electrophilic chlorine atom attaches itself to the bare
electron pair of nitrogen, thus releasing water. Conversely,

16
the reaction may be viewed as one of displacement whereby the
nucleophilic amine displaces OH’ from H0C1 (Morris, 1978) .
Amides also appear to react via the same pattern or via the
haloform reaction but at much slower rates. A simplified
scheme of chlorine substitution with organic N-substituted
organic compounds is shown below (Morris, 1967):
R-NH2 + H0C1
> R-NHC1 +
h2o
15.
R-CO-NH2 + H0C1 —
> R-C0-NHC1
+ h2o
16.
In both cases,
the rates of
chloramination
vary
proportionately with the basic strength or nucleophilicity of
the nitrogenous substrate (Morris, 1978). Proteins are more
complex than amines and amides and chlorination of organic
nitrogen from proteinaceous material is a slow and tedious
process. However, proteins may also undergo substitution with
chlorine to form N-chlorinated proteinaceous byproducts
(Jolley et al., 1978; White, 1972).
Formation of C-chlorinated compounds. Formation of
C-chlorinated compounds via chlorine substitution is perhaps
the most widespread in nature (Jolley et al., 1978; Morris,
1975; Carlson et al. , 1975). The major substitution reactions
of chlorine involve substitution into aromatic or heterocyclic
compounds and the haloform reaction. Humic and fulvic
substances abound in nature and represent vegetation under
varying degrees of decay. These complex and otherwise poorly
characterized high molecular weight materials contain aromatic
and heterocyclic moeities which readily undergo chlorination

17
reactions in aqueous chlorine solutions when activated (Jolley
et al., 1978).
Some substitution reactions involving phenolic compounds
and aromatic organic acids are presented in Figure 1. As
before, the mechanism of the reaction involves an
electrophilic attack of H0C1 on the phenoxide ion. In this
case the reaction is facilitated by the presence of a negative
charge on the nucleophilic substrate and only when the ring
is "activated" by substituents such as the oxide ion, does
substitution occur (Morris, 1978). Phenols are more reactive
in dilute aqueous chlorine solutions than other aromatic
compounds possessing different substituent groups (Carlson and
Caple, 1978) . Substitution may proceed at the activated ortho
or para positions not fully occupied by chlorine atoms.
Further rupture of the aromatic ring may occur by mechanisms
that are not fully understood (Morris, 1978). Heterocyclic
aromatic compounds such as the nucleic acid bases may also
undergo substitution reactions in which they are either
activated or inactivated toward electrophilic attack compared
to benzene (Morris, 1978). An example of the activation
process involves the chlorination of some purines and
pyrimidines. Aqueous chlorination of the pyrimidines,
cytosine and uracil has been shown to lead to the formation
of their corresponding 5-chloro analogs (Figure 2) and other
poorly characterized byproducts (Jolley et al., 1975? Hoyano
et al., 1973; Patton et al., 1972).

18
COOH
OH
+
HOH
COOH
+ HOCI
COOH
HOH
Figure 1. Chlorine substitution reactions with some phenolic
compounds and aromatic organic acids (Adapted from
Jolley et al., 1978).

19
Figure 2
NH.
NH.
N
cAn
+ HOC!
N
\k
C!
+ HOH
H
0
/'-N'
H
0
1!
HN
u H
4- HOC!
0
ii
( HN
H
Cl
+ HOH
0
li
HN
0
'' ^N
N
LAJ
+ HOCI
H
N'
H
0
ii
HN^
»"Sf
N
H
N
J-
C!
+ HOH
N
Ov^N^ ^N
ch3 H
ch3
i J
N
+ HOCI
N
OH
ch3
I J
N
CH
Chlorine substitution reactions with pyrimidines
and purines (Adapted from Jolley et al., 1978).

20
Similar chlorinated products may be obtained with purines
as shown in Figure 2. With the purines, actual formation of
the chlorinated analogs has not been demonstrated in dilute
aqueous chlorine solutions but rather in non-polar solvents
and in acetic acid (Jolley et al., 1978). Sources of nucleic
acid bases in potable water are plankton, bacteria, and plant
and animal matter. Like the nucleic acid bases, pyrroles are
also thought to be greatly activated to undergo chlorine
substitution reactions (Morris, 1978). Pyridines on the other
hand are thought to be sufficiently deactivated so that
reaction with dilute aqueous chlorine solutions is not
expected to occur in the ring (Morris, 1978) .
Finally, formation of C-chlorinated compounds may occur
through the haloform reaction (Figure 3) with humic material
leading to the formation of volatile compounds such as
chloroform (Morris, 1975; Rook, 1974).
Chlorine disinfection
Despite much research done in this area, the actual
mechanism of germicidal action of chlorine has not been fully
elucidated. It is well known that even in trace amounts,
chlorine has fast killing action on bacteria and several
theories have been proposed to explain this killing action.
To date, none of the theories has been fully accepted due to
a lack of substantive evidence (Dychdala, 1983; White, 1972).
The proposal that the germicidal action of chlorine is due to
the liberation of nascent oxygen held for some time until it

21
Figure 3. Detailed reaction mechanism of the haloform
reaction. X=Halogen (Adapted from Morris, 1985).

22
was finally dispelled. This is because other strong nascent
oxygen producers such as hydrogen peroxide and potassium
permanganate are less effective bactericides than chlorine
(White, 1972). Also, chlorine still possesses germicidal
activity under conditions which exclude direct oxidation of
bacterial protoplasm (Mercer and Somers, 1957). Other
theories, even though attractive, have not received the full
support of researchers in this area. Baker (1926) proposed
that chlorine reacted with bacteria cell membrane proteins to
produce N-chloro compounds which interfere with bacterial cell
metabolism leading to death. The N-chloro theory was taken
a step further by Rudolph and Levine (1941) who concluded that
the bactericidal activity of chlorine or hypochlorites
proceeds in two stages: 1) the penetration of an active
ingredient into the bacterial cell, and 2) the chemical
reaction of this ingredient with the cellular protoplasm to
form toxic N-chloro compounds which destroy the organism.
Mercer and Somers (1957) stated that bacterial death by
chlorine is a poisoning process in which a form of chlorine
combines chemically with the protoplasm of the bacterial cell
to produce toxic organic complexes.
Other proposed mechanisms of chlorine disinfection were
directed at key enzymes in the target organisms. Green and
Stumpf (1946) and later Knox et al. (1948) observed
experimentally that there was a correlation between the effect
of chlorine on bacterial growth and its effect on the rate of

23
glucose oxidation by certain bacteria. They were able to show
that a bacterial suspension became sterile when the organisms
lost their ability to oxidize glucose. The mechanism of
inhibition was thought to involve irreversible oxidation of
the sulfhydryl groups of essential enzymes like
triosephosphate dehydrogenase. The sulfhydryl oxidation
theory received additional support when chlorine was shown to
oxidize sulfhydryl radicals to sulfonyl compounds and sulfides
to sulfates under conditions that simulate water chlorination
(Ingols et al., 1953; Black and Goodson, 1952; Douglas and
Johnson, 1938). Wyss (1962) postulated the theory of
unbalanced growth in which destruction of part of the enzyme
system throws the bacteria out of metabolic balance leading
to death. Studying the effect of chlorine on bacterial
enzymes, Venkobachar and coworkers (1975) observed that in the
bactericidal concentration of chlorine total dehydrogenase
activity in Eschericia coli was markedly affected and the
inhibition correlated with percent bacteria kill. They also
observed that succinyl dehydrogenase activity in the bacteria
was inactivated probably due to the oxidation of sulfhydryl
groups at lower chlorine doses.
Using radiolabelled chlorine, Friberg (1956) observed
that the first contact oxidation reactions of chlorine with
bacterial cells was responsible for bacterial death and not
through chloramine formation. He later demonstrated that
chlorine, even in minute amounts, reacts with and results in

24
destructive permeability changes in bacterial cell walls
resulting in death (Friberg, 1957). Additional mechanisms of
chlorine disinfection have been reported to include induction
of chromosomal aberrations (Ingols, 1958); disruption of
protein synthesis (Bernade et al., 1967); reaction with
purines and pyrimidines of nucleic acids (Hoyano et al.,
1973); oxidative decarboxylation of amino acids (Pereira et
al., 1973) ; loss of macromolecules due to inhibition of oxygen
uptake and oxidative decarboxylation (Venkobachar et al.,
1975); production of lesions in DNA (Shih and Lederberg,
1976); inhibition of respiration and active nutrient transport
(Camper and McFeters, 1979); disruption of bacterial spore
coats and increasing spore permeability (Foegeding, 1983).
Chlorine has also been reported to inactivate poliovirus by
reacting with the outer protein coat of the virus (Tenno et
al., 1980) or by damaging the internal RNA (Olivieri et al.,
1980; O'Brien and Newman, 1979).
Of the active chlorine species, hypochlorous acid is by
far the most active disinfectant. Its germicidal efficiency
is related to the ease with which it can penetrate cell walls.
The ease of cell penetration is comparable to that of water
which is structurally similar. Also its relatively low
molecular weight and electrical neutrality allow easy cell
penetration (White, 1972). The germicidal efficiency of free
available chlorine depends on other factors such as pH,
temperature, chlorine concentration, length of contact time,

25
and type and concentration of microorganisms. The pH of the
solution is perhaps the most important of all the above
factors because an increase in pH causes a substantial
decrease in biocidal efficiency and vice versa. This is
because at higher pH, hypochlorous acid is converted to
hypochlorite ion (equation 2) which is an inferior biocide.
The hypochlorite ion obtained from hypochlorous acid
dissociation is a relatively poor disinfectant because of its
inability to diffuse through microbial cell walls. The
inability to traverse microbial cell walls is due to the
negative charge associated with it (White, 1972). Combined
available chlorine as chloramines are also less effective
bactericidal agents than chlorine. Chloramines are slow-
acting and require more than a hundred-fold increase in
contact time and much higher concentrations to achieve the
same bacterial lethality as chlorine (Kabler, 1953;
Butterfield, 1948).
Chemistry of Chlorine Dioxide
Chlorine dioxide is a polar gas that readily dissolves
in but does not react with water to produce aqueous chlorine
dioxide. It is a true dissolved gas because it does not react
with water (Masschelein, 1979). The basic reaction of Davy,
who discovered the gas by pouring a strong acid over sodium
chlorate in 1911, is still used today (White, 1972):
NaC103 + 2HC1 > Cl02 + NaCl + H20 + Cl 17.

26
Chlorine dioxide has an intense greenish yellow color with a
distinctive odor similar to chlorine but more irritating and
more toxic (White, 1972). One important physical property of
chlorine dioxide is that it is five times more soluble in
water than chlorine. It is extremely volatile and decays by
photodecomposition, volatilization and decay reactions. In
fact, it can easily be removed from agueous solutions merely
by aeration. It is very unstable and explosive and as such,
cannot be transported but is usually prepared on site (White,
1972) .
Chlorine dioxide does not belong to the family of
"available chlorine" compounds, i.e., those compounds that
hydrolyze to form hypochlorous acid, but due to its oxidizing
power it is referred to as having an available chlorine
content of 263 percent. It has a larger oxidation capacity
than H0C1 because it can accept 2.5 times more electrons than
H0C1 (Ingols and Ridenour, 1948). This can be deduced from
reactions involving iodine liberation from iodide in the
iodometric titration method:
C102 + 51' + 4H+ > Cl' + 2.5I2 + 2H20 18.
H0C1 + 21" > Cl' + I2 + OH' 19.
The oxidation potential of chlorine dioxide is less than
that of H0C1 as indicated by its redox potential of 0.95V
compared to that of H0C1 in eguation 4. The redox potential
of chlorine dioxide and chlorite are presented below
(Masschelein, 1979):

27
C102 + e' > C102' (0.95V) 20.
C102' + 2H20 + 4e" > Cl' + 40H' (0.78V) 21.
The oxidizing capacity of chlorine dioxide is not all used in
water treatment because in practice chlorine dioxide is
reduced to chlorite according to equation 20. The chlorite
ion is an effective oxidizing agent and is consumed at a much
slower rate in oxidation-reduction reactions.
Reaction with inorganics
In the treatment of drinking water, chlorine dioxide is
commonly used for the removal of divalent ferrous iron and
manganese and sometimes sulfide through oxidation reactions
(White, 1972). Much higher temperatures are required for
oxidation of trivalent chromium salts to chromate and will
not be discussed here (Masschelein, 1979).
Oxidation of iron. In water treatment, an effective means of
iron removal is through chlorine dioxide oxidation of iron
from the moderately soluble ferrous (II) to the ferric (III)
state as an insoluble ferric hydroxide precipitate which can
be removed by filtration. The above reaction is presented in
equation 22.
C102 + FeO + NaOH + H20 > Fe(0H)3 + NaC102 22.
The pH optimum for the above reaction is above 7.0, and is
preferable in the range between pH 8-9. The reaction is a
slow one, and in most cases, chlorine is the chemical of
choice for this type of reaction. This is because chlorine
is cheaper and equally effective as chlorine dioxide (White,

28
1972). Chlorine dioxide has been shown to oxidize organically
bound iron (Masschelein, 1979).
Oxidation of manganese. Manganese is present in many wells
and surface waters and can cause blackness of water at high
concentrations. Even though it is not known to cause any
health problems, the maximum level permitted in water is 0.05
mg/L (Knocke et al., 1987). Chlorine is used for manganese
removal but the reaction is too slow to be desirable.
Chlorine dioxide reacts more rapidly with manganese compounds
than chlorine. In this reaction, manganese (II) is oxidized
to manganese (IV) as insoluble manganese dioxide according to
the following reaction (White, 1972):
2C102 + MnS04 + 4NaOH > Mn02 + 2NaCl02 +
Na2S04 + 2H20 23.
The stoichiometric dosage of chlorine dioxide required for
manganese oxidation has been established as 2.45 mg/mg Mn2+
assuming the reduction byproduct is chloride. However, under
laboratory conditions, much higher doses of chlorine dioxide
are required. Knocke and coworkers (1987) have recently shown
that at least twice the stoichiometric amount of chlorine
dioxide was required to lower manganese levels to less than
0.05 mg/L. Also, when the total organic carbon content of the
water was high, much more chlorine dioxide was required than
permitted by law to limit manganese content to acceptable
levels.

29
Optimum conditions for the reaction have not been fully
elucidated but it is thought that complete oxidation of
manganese occurs at pH values higher than 7 (White, 1972).
Like iron, chlorine dioxide will oxidize organically bound
manganese. The chlorite formed from chlorine dioxide
reduction can also be used to reduce manganese or iron under
similar conditions (Masschelein, 1979).
Oxidation of sulfides. Hydrogen sulfide is found dissolved
in underground waters and has been shown to react with
chlorine to produce sulfates and elemental sulfur. Chlorine
dioxide oxidation of hydrogen sulfide to sulfate occurs
extensively in the pH range 5-9 (White, 1972). The type of
products formed, either elemental sulfur or sulfate, is
dependent on pH and temperature, with higher temperatures and
pH favoring sulfate formation (Dohnalek and FitzPatrick,
1983) . Reactions of chlorine with sulfur has fully been
investigated and characterized but the same does not hold true
for chlorine dioxide. Until more data becomes available on
chlorine dioxide oxidation of sulfides, chlorine remains the
chemical of choice for sulfide removal since it is cheaper and
data is available on the type of byproducts likely to be
encountered.
Reaction with organics
Chlorine dioxide is a selective oxidant which reacts with
organic matter via oxidation to produce both volatile and
non-volatile reaction products. In some cases, however, some

30
chlorinated compounds are encountered. This type of
chlorination reaction is thought to be an indirect one and
probably corresponds to a progressive reduction of the dioxide
passing through the hypochlorous acid stage (Masschelein,
1979) . These chlorination reactions involving chlorine
dioxide arise presumably due to the presence of chlorine as
a contaminant in the dioxide used (Masschelein, 1979) .
Chlorine dioxide does not react with ammonia to form
chloramines; therefore, chloramine toxicity is avoided with
its use. Unsubstituted aromatics, primary amines and many
other organic compounds are unreactive towards chlorine
dioxide (Raugh, 1979).
Oxidation of phenols and odor control. Phenols are ubiquitous
pollutants in water and the environment and have been used as
model compounds for chlorine reactions and haloform formation
(Aieta and Berg, 1986). Oxidation of phenols and phenolic
compounds by chlorine and chlorine dioxide has been
extensively investigated because phenolic tastes and odors are
produced in natural waters as a result of industrial waste
pollution of raw water. Petroleum and wood processing plants,
algae and decaying vegetation are principal sources of
phenolic compounds. Chlorination of polluted waters leads to
the formation of chlorophenols such as 2-chlorophenol ,
2,4-dichlorophenol, 2,4,6-trichlorophenol and others which
possess distinct offensive tastes and odors (White, 1972) .

31
Chlorine dioxide has been shown to be specific for the
control of these phenolic and chlorophenolic tastes and odors
many times faster than free available chlorine. This is
related to the high oxidizing capacity of chlorine dioxide
when compared to chlorine (Masschelein, 1979; White, 1972).
Apart from control of phenolic tastes and odors, chlorine
dioxide has successfully been used against algae odors, fishy
tastes, and earthy-musty taste and odor compounds (Lalezary
et al., 1986; Wright, 1980; Medker et al., 1968).
Reaction of egual parts of chlorine dioxide with phenols
has been reported to yield p-benzoquinone and 2-chloro-
benzoquinone in the concentration range 1-1000 mg phenol/L.
Organic acids, mainly maleate and oxalate are also formed if
the ratio of chlorine dioxide to phenol is increased five-fold
(Wajon et al., 1982; Masschelein, 1979). Extended oxidation
with excess chlorine dioxide leads to the formation of
carboxylic acids and, under moderate conditions, quiñones.
When excess phenols are used, chlorophenols are formed due to
the slow release of hypochlorous acid which subsequently
reacts with excess phenol (Wajon et al., 1982). Under water
treatment conditions, the chlorine dioxide to phenol ratio
will be greater than unity and the major products likely to
be encountered are p-benzoquinone and simple organic acids.
Other oxidative reactions. Carbohydrates, proteins and amino
acids and other food components have been shown to undergo
mainly oxidative reactions with chlorine dioxide and are

32
discussed later in this chapter. Environmentally significant
compounds such as polynuclear aromatic hydrocarbons (PAHs) and
their heterocylcic analogs also react with chlorine dioxide
to varying degrees. Lin and Carlson (1984) subjected some
environmental heterocycles to dilute aqueous solutions of
chlorine, chloramine and chlorine dioxide and the latter was
found to produce mainly oxygenated byproducts. For example,
chlorine dioxide oxidation of indole produced oxindole and
isatin.
Chlorination reactions. As mentioned earlier, chlorination
or chlorine incorporation is not an intrinsic reaction of
chlorine dioxide. These reactions are secondary reactions
which are thought to occur through the release of hypochlorous
acid from a primary product or from impurities in the chlorine
dioxide (Masschelein, 1979). In reactions involving chlorine
and chlorine dioxide incorporation into lipids, Ghanbari et
al. (1982b) showed that chlorine incorporation was many orders
of magnitude higher with chlorine than chlorine dioxide.
Chlorine dioxide disinfection
Even though the bactericidal properties of chlorine
dioxide were known at the beginning of this century, the
practical uses of said properties were not fully utilized
until the middle of the century. This is because chlorine
dioxide was first applied to water treatment around the middle
of the century and also increased environmental and water
pollution led to a search for alternate and varied

33
disinfectants (Masschelein, 1979 ; White, 1972). McCarthy
(1944) first recommended the use of chlorine dioxide for the
disinfection of water of low organic content. This is because
increasing the organic content of water decreased the
germicidal effectiveness of chlorine dioxide. Ridenour and
Ingols (1947) showed that chlorine dioxide was as effective
as chlorine for the control of E. col i and other bacterial
populations. These workers were the first to show that unlike
chlorine, the bactericidal efficiency of chlorine dioxide was
unaffected over a wide pH range (from pH 6-10), which makes
it suitable for the disinfection of high pH waters. On the
other hand, the germicidal effectiveness of chlorine dioxide
was found to decrease at low temperatures (Ridenour and
Ambruster, 1949).
Most of the studies regarding chlorine dioxide
disinfection have focussed on a comparison with that of
chlorine and, as a result, very little information is
available on its mode of action. Some of the comparative
studies have shown that chlorine dioxide is a more efficient
virucide and bactericide than chlorine (Alvarez and O'Brien,
1982; Aieta et al., 1980; Longley et al. , 1980; Sawyer, 1976).
The proposed mechanisms of action of chlorine dioxide on
microbes are similar to those of chlorine; either through a
reaction with macromolecules or through gross physiological
changes. Like chlorine, there is evidence that chlorine
dioxide acts on dehydrogenase enzymes located in bacterial

34
cytoplasmic membranes but this evidence has not been
substantiated.
Chlorine dioxide is reactive towards several compounds
including phenols, some amino acids and proteins and this
reactivity has been used to explain its bactericidal action.
Fujii and Ukita (1957) demonstrated that chlorine dioxide
oxidation of tryptophan produced some indole derivatives of
tryptophan which may explain the bactericidal action of
chlorine dioxide. On the mode of killing action of chlorine
dioxide on bacteria, Bernade and coworkers (1967) concluded
that chlorine dioxide was sufficiently reactive with several
amino acids to alter their structures and thus, react with
cell walls. They also observed that protein synthesis was
abruptly inhibited by the action of chlorine dioxide. Roller
et al. (1980) have reported that under laboratory conditions,
the level of chlorine dioxide used in treatment plants does
not affect DNA or protein synthesis. Contrary to the above,
Alvarez and O'Brien (1982) in their studies on poliovirus
concluded that chlorine dioxide reacted with viral RNA leading
to virus inactivation.
Noss and others (1983) investigated the reactivity of
chlorine dioxide with nucleic acids, proteins and amino acids.
Chlorine dioxide readily and rapidly reacted with the amino
acids cysteine, tyrosine and tryptophan but not with viral
RNA. The authors postulated that the virucidal activity of
chlorine dioxide was probably due to alteration of viral

35
proteins. Further reports by the same authors suggest that
chlorine dioxide reaction with tyrosine appears to be the main
mechanism of action of F2 virus inactivation by chlorine
dioxide (Olivieri et al., 1985).
Another probable mechanism of action of chlorine dioxide
may be due to its interaction with membrane lipids and fatty
acids leading to permeability changes in membranes. Ghanbari
et al. (1982b) showed that chlorine dioxide was highly
reactive towards free fatty acids leading to the formation of
mainly oxidized byproducts. The authors observed that free
fatty acids were more reactive with chlorine dioxide than
their methyl esters. From the foregoing discussion, it is
possible that chlorine dioxide oxidation of membrane lipids
and proteins leads to microbial death. Even though some of
the reactions have been reported to occur in model systems,
the actual mechanisms that take place in the intact
microorganisms leading to death still need to be proven.
Reactions of Aqueous Chlorine with Amino Acids. Peptides
and Proteins
Proteins are complex high molecular weight constituents
of plant and animal matter made up of carbon, nitrogen,
oxygen, hydrogen and, in some cases, sulfur. Of these
elements, carbon, nitrogen and sulfur exert a chlorine demand
(White, 1972). Proteins and protein material from many
sources contribute greatly to the organic nitrogen content of

36
natural waters and pose a problem during water chlorination
(Qualls and Johnson, 1985). Amino acids are water soluble and
are typically found in natural waters at concentrations in the
range 2-400 jig/L (Le Cloirec and Martin, 1985) . During water
treatment, the amount of amino acids can increase
substantially due to hydrolysis-depolymerization of proteins
or peptides, or due to microbial metabolism (Le Cloirec and
Martin, 1985) . In the food industry, amino acid
concentrations are likely to be much higher in process
cleaning waters, and superchlorination of process waters is
sometimes used to control microbial contamination (Robinson
et al., 1981).
The reactions of chlorine with amino acids, proteins and
peptides have been studied in detail and reported to produce
both oxidized and chlorinated byproducts under different
treatment conditions (Jolley et al., 1978 ; Pereira et al. ,
1973). The first report in the literature on the chlorination
of amino acids was made by Langheld (1909) who reported that
sodium hypochlorite reacted with alpha-amino acids to form
mono- or dichloroamino acids which subsequently decomposed to
form aldehydes. Langheld (1909) observed that the rate of
monochloramine decomposition was affected by the amino group;
primary amino groups decomposed to give aldehydes or ketones
as well as ammonia while secondary amino groups produced
amines instead of ammonia. Dakin et al. (1916) and Dakin
(1916) confirmed Langheld's study and showed that nitriles

37
were formed as major products when excess chlorine was used.
Dakin and colleagues showed that hydrogen cyanide was produced
from glycine, acetonitrile from alanine, isobutyl cyanide from
leucine and cyanobenzene from phenyl acetate. The general
mechanism proposed by Dakin (1916) involves the initial
formation of a dichloroamino acid which undergoes further
decomposition to form a cyanide. The chlorination of several
amino acids including those studied by Dakin and coworkers has
been further investigated (Isaac and Morris, 1983a; Margerum
et al., 1979). These studies showed that at neutral pH all
the amino acids tested readily reacted with chlorine with the
possible involvement of N-chloro compounds as intermediates.
Rydon and Smith (1952) developed a paper chromatographic
method for determining N-chloroamino acid formation during
chlorination. They showed that several amino acids including
glycine, alanine, leucine, serine, threonine, lysine,
phenylalanine and histidine liberated chlorine from the
corresponding N-chloro derivatives which reacted positively
with a starch-potassium iodide spray. Cysteine, cystine,
methionine and tyrosine failed to release chlorine due
probably to the formation of mainly oxidized products.
The importance of pH in product formation was
investigated by Wright (1936) who observed that aldehydes were
formed from alpha-amino acids on reaction with sodium
hypochlorite at neutral pH while nitriles predominated at
lower pH. From his point of view, acidity favors chlorination

38
and alkalinity favors oxidation. Wright showed that under
acidic conditions glycine was first chlorinated, and the
resulting chloroamino acid was then oxidized by any available
chlorine present; whilst under alkaline conditions,
destruction of available chlorine was mainly due to oxidation.
Jolley and Carpenter (1983) also observed that at neutral pH,
monochlorinated amino acids were formed with aqueous chlorine,
while dichloro derivatives were produced as major products
under acidic conditions. Stanbro and Smith (1979) studied the
kinetics and mechanisms of decomposition of N-chloroalanine
in aqueous solution and showed that the decomposition products
included acetaldehyde, ammonia and carbon dioxide and,
depending on pH, pyruvate. Le Cloirec and Martin (1985) have
provided additional evidence to support the general mechanisms
already proposed. A summary of the general reaction
mechanisms leading to the formation of nitriles and aldehydes
(Figure 4) has been provided by Le Cloirec and Martin (1985).
In the reactions leading to the formation of aldehydes
and nitriles, imine-type compounds are known as essential
intermediates but have not been isolated in the chlorination
of individual amino acids. However, in the case of a peptide,
DL-alanyl-DL-leucine, an imine-type compound has been isolated
and characterized (Pereira et al., 1973). Pereira et al.
(1973) also showed that nitriles are major and aldehydes are
minor products of amino acid chlorination reactions. Tan et
al. (1987a) studied the kinetics of the reactions of aqueous

39
COOH
| HOC1
R.CH
I
nh2
Figure 4.
COOH
| -HC1 -HC1
R.CH > R.CH = NCI > R. C — N
I -co2
NC12 Nitrile
A
HOC1
COOH
| -C02 HOC1
R.CH > R.CH = NH >
| -HCl
NHC1
OH
| -nh2ci
R. CH > RCHO
I
NHC1 Aldehyde
Reactions of chlorine with amino acids leading to
the formation of nitriles and aldehydes (Adapted
from Le Cloirec and Martin, 1985)

40
chlorine and several amino acids in different buffers and
observed that all the amino acids were reactive towards
chlorine over a wide pH range. The type of buffer system was
found markedly to affect the reaction rates. These workers
did not identify any of the reaction products but confirmed
previous reports (Isaac and Morris, 1983a) that the reaction
rates of the sulfur-containing amino acids, cysteine and
methionine, as well as tryptophan were too rapid to be
monitored by the iodometric method of titration. Jancangelo
and Olivieri (1985) have also reported that the sulfur-
containing amino acids and the heterocyclic amino acid,
tryptophan, are the most reactive with monochloramine.
The specific functional groups of the amino acids have
been shown to affect the type of products likely to be formed
during chlorination. In the case of the sulfur amino acid,
cysteine, Pereira and colleagues (1973) have shown that the
major products of chlorination were cystine and cysteic acid
while cystine produced only cysteic acid as the end product.
Amino acids with phenyl or heterocyclic groups readily form
chlorinated analogs if the rings are sufficiently activated.
For example tyrosine and tryptophan have been shown to form
the corresponding mono- and dichloro derivatives upon aqueous
chlorination (Trehy et al., 1986; Burleson et al., 1980;
Pereira et al., 1973). Aqueous chlorination of tyrosine has
been reported to yield mono- and dichlorobenzeneacetonitrile
and mono- and dichlorobenzeneacetaldehyde (Trehy et al., 1986;

41
Burleson et al., 1980; Pereira et al., 1973). Major
chlorination products of phenylalanine were previously
reported to be phenylacetonitrile (95%) and phenylacetaldehyde
(5%) but some chlorinated derivatives have been identified as
well (Horth et al., 1987).
In the case of tryptophan, Kirk and Mitchell (1980)
observed the formation of a dark-colored precipitate when
reacted with different concentrations of aqueous chlorine.
Although the reaction products were not fully identified, the
authors isolated and partially characterized a monochlorinated
product. Using structurally similar indole compounds, they
showed that the intact heterocyclic ring was very important
in color formation. Such color changes have been used
colorimetrically to determine chlorine dioxide concentrations
in water upon reaction with tyrosine (Hodgen and Ingols,
1954). Tryptophan has also been reported to produce oxidized
indole derivatives as well as ether extractable chlorinated
analogs (Owusu-Yaw et al., 1989; Trehy et al., 1986).
Nitriles and aldehydes are also produced as a result of
aqueous chlorination of tryptophan (Owusu-Yaw et al., 1989;
Horth et al., 1987).
The products of the reactions of chlorine with peptides
and proteins have been reported to be mainly N-chloro or
N,N-dichloro derivatives (Pereira et al., 1973, Rydon and
Smith, 1952). Rydon and Smith (1952) showed that peptides
and proteins reacted with chlorine gas to form

42
N-chloropeptides which could be detected visually on paper
chromatograms using a starch-potassium iodide spray.
According to the authors, color was developed from the
liberation of chlorine from the N-chloropeptide bond which
reacted with the starch iodide-indicator. Pereira and
coworkers (1973) investigated the reactions of excess chlorine
with the four dipeptides, glycyl-L-alanine, glycyl-DL-
phenylalanine, DL-alanine-DL-leucine and L-valyl-L-valine and
showed that the corresponding N,N-dichloro-dipeptides were the
major reaction products. From their point of view, the amide
nitrogen of the peptide was not attacked, and the peptide bond
itself was relatively resistant to chlorination. These
authors were the first to show that N-chloroimines were
produced by the dehydrochlorination of the N,N-dichloro-
dipeptide DL-alanyl-DL-leucine with ethanolic sodium hydroxide
at ambient temperature.
Glycylglycine has been used as a model for the formation
of N,N-dichloroglycylglycine and N-chloroglycylglycine (Isaac
and Morris, 1985; Qualls and Johnson, 1985; Margerum et al.,
1979). These studies show that the rate of chlorination of
the dipeptide is faster than that of several amino acids and
ammonia and that the peptide will be preferentially
chlorinated over ammonia or the amino acids glycine, alanine,
serine and glutamate in water. Other peptides such as
phenylalanylglycine and glycylglycylalanine have been reported
to produce stable chloramine residuals (Horth et al. , 1989).

43
From all the studies cited, it becomes clear that the fastest
initial reactions of peptides with chlorine occur at the
nucleophilic terminal amino groups or the reactive side groups
of amino acids. For example when flour was chlorinated with
excess chlorine gas, the levels of the amino acids,
methionine, threonine, cysteine and histidine in the proteins
decreased by approximately, 68, 41, 32 and 17%, respectively
(Ewart, 1968).
Tan et al. (1987a) studied the kinetics of the reactions
of aqueous chlorine and three peptides and observed that the
rates of reaction of L-glycyl-L-tryptophan and L-tryptophyl-
glycine were too rapid to be measured iodometrically. The
authors suggested that the rapid reaction rates were due
mainly to the reaction of chlorine with the heterocyclic ring
of tryptophan and not with the amino or carboxyl groups. In
the case of aspartame, even though the component amino acids,
aspartate and phenylalanine, were individually reactive
towards chlorine, the dipeptide was not. The authors did not
provide any conclusive evidence as to whether any of the
reaction products were chloramines. Apart from the formation
of N-chloro compounds, Tsen and Kulp (1971) have postulated
that amino acid side chains of proteins are progressively
cleaved by increasing concentrations of chlorine. They
observed that the amount of water-extractable proteins in
chlorinated flour increased as the chlorine concentration was
increased. Chlorine has also been shown to be readily

44
incorporated into proteins from different sources including
shrimp, meat and poultry (Johnston et al, 1983; Cunningham and
Lawrence, 1977); egg albumin, gelatin, casein, and bovine
serum albumin (Baker, 1947; Wright, 1936 and 1926; Milroy,
1916); and flour proteins (Wei et al., 1984).
Reactions of Chlorine Dioxide with Amino Acids.
Peptides and Proteins
Chlorine dioxide has been proposed as an alternate to
chlorine for disinfection purposes and it is therefore
important to understand the kind of reactions and byproducts
likely to be formed with its use, especially proteinaceous
material. The reactions of chlorine dioxide under water
treatment conditions are mainly oxidative and as such, it has
been used for the control of phenolic tastes and odors in
natural waters (White, 1972). Several reports are available
in the literature on the reactions of chlorine dioxide with
amino acids under different conditions, but as pointed by
Masschelein (1979), the conditions of the reaction are very
important.
In earlier investigations, Schmidt and Braunsdorf (1922)
observed that most amino acids were not reactive towards
chlorine dioxide and that the least reactive were glycine,
leucine, serine, alanine, phenylalanine, glutamate,
asparagine, valine and hydroxyproline. The authors found
cystine to be very reactive. Bernade et al. (1967) further

45
investigated the reaction of chlorine dioxide with several
amino acids and observed that after a contact time of 3 0
minutes, asparagine, histidine, phenylalanine, arginine,
proline and leucine were unreactive. Contrary to the above
findings, Kennaugh (1957) noted that when a mixture containing
tryptophan, tyrosine, proline, threonine, methionine, valine
and lysine were mixed with diaphanol, a solution of 50% acetic
acid saturated with chlorine dioxide, the aromatic amino acids
disappeared after 18 hours. Also, most of the amino acids
were found to react after 3 days and phenylalanine did not
produce any ninhydrin positive material. Noss et al. (1983)
studied the reactivity of equimolar concentrations (2.5 x
10"4 M) of twenty-two amino acids and four nucleic acids with
chlorine dioxide at neutral pH and found only seven (cysteine,
cystine, proline, histidine, hydroxyproline, tyrosine and
tryptophan) to be reactive. Additional evidence in support
of these studies was provided by Tan et al. (1987b) who showed
that six of twenty-one amino acids were reactive with chlorine
dioxide. Of the six, the reactions of cysteine, tryptophan
and tyrosine were too rapid to be measured by the iodometric
titration method.
From the above and other studies, it is clear that the
reactivity of chlorine dioxide with amino acids and proteins
depends on pH, temperature and contact time, with higher
temperatures and contact times and lower pH favoring the
reaction. Also, specific functional groups of the amino acids

46
and proteins such as phenyl or sulfhydryl and the relative
molar concentrations of reagent and substrate affect the
reactions. For example, glycine and phenylalanine were inert
toward chlorine dioxide at room temperature when equimolar
concentrations of reagent and substrate were used (Noss et
al. , 1983) . However, the reactions were found to proceed when
excess molar concentrations of chlorine dioxide were used
(Taymaz et al., 1979).
The sulfur-containing amino acids and those with phenyl
or heterocyclic rings have been reported to be the most
reactive with chlorine dioxide (Masschelein, 1979) . Of the
amino acids containing sulfur, cystine has been reported to
produce cysteic acid with a cystine disulfoxide intermediate.
The reaction is said to involve the breakage of the sulfur-
sulfur bond of cystine followed by further oxidation
(Masschelein, 1979) . Under similar conditions, methionine is
oxidized to the sulfoxide and then to the sulfone. Other
organic sulfides and mercaptans are also known to undergo
similar oxidative reactions with chlorine dioxide.
Chlorine dioxide oxidation of tyrosine is similar to
those of phenols which are especially susceptible to
oxidation. When pure chlorine dioxide is used, no chlorinated
products are obtained (Masschelein, 1979) . With chlorine
dioxide, tyrosine has been reported to produce pink
dopaquinone and red dopachrome. This reaction has been used
as a basis for the colorimetric determination of chlorine

47
dioxide concentrations in water (Hodgen and Ingols, 1954).
When excess chlorine dioxide, 3 moles per mole of tyrosine,
is used for an extended period of time, other unidentified
colored products are formed as well. Reaction of 2 molars
excess of chlorine dioxide with phenylalanine at elevated
temperatures has been reported to produce phenylacetic acid,
phenylacetaldehyde, benzoic acid, benzaldehyde and mandelic
acid (Taymaz et al., 1979). No chlorinated products were
identified from the above reaction. When the chlorine dioxide
concentration was increased to 10 excess molar under similar
conditions, no phenylalanine or above-mentioned products were
detected after 6 days. From the foregoing, it is possible
that further oxidation of the initial reaction products takes
place with excess chlorine dioxide thus giving off carbon
dioxide and causing the disappearance of substrate.
The heterocyclic group of compounds is also very
susceptible to chlorine dioxide oxidation leading to the
formation of complex oxidized products. Chlorine dioxide
oxidation of tryptophan produced indoxyl, isatin, indigo red
as well as unidentified polymerization products (Fujii and
Ukita, 1957). Structurally similar indole derivatives such
as indole itself, 3-methyltryptophan, 3-indolelactic acid and
3-methylindole have also been shown to be readily oxidized by
chlorine dioxide, sometimes leading to color and precipitate
formation (Sen et al., 1989; Lin and Carlson, 1984). Indole
is oxidized to oxindole and isatin while 3-methylindole

48
produces 3-methyloxindole and o-formamidoacetophenone (Lin and
Carlson, 1984) .
Unlike amino acids, the reaction of chlorine dioxide with
proteins and peptides has not received much attention in model
systems. A review of the available literature has been
documented by Fukayama et al. (1986). Tan et al. (1987b)
studied the kinetics and mutagenicity of three peptides with
chlorine dioxide at room temperature using equimolar
concentrations of reagent and substrate. They observed that
the reactions of two dipeptides L-tryptophyl-glycine and
L-glycyl-L-tryptophan were too fast to be determined by
iodometric titration. They attributed the very fast reaction
rates to the presence of the tryptophan moieties which have
previously been shown to react rapidly with chlorine dioxide
(Fujii and Ukita, 1957). The third dipeptide, aspartame, was
found to be relatively unreactive since less than 10% of the
initial reagent was lost after 2 hours of reaction. The
authors explained that the inertness of this dipeptide with
chlorine dioxide could be attributed to the component amino
acids, aspartate and phenylalanine, which were individually
unreactive towards chlorine dioxide.
Chlorine gas and chlorine dioxide have traditionally been
used to treat flour to improve baking properties and dough
quality. Meredith et al. (1956) treated wheat flour with high
levels of chlorine dioxide and did not find any chlorinated
derivatives of amino acids and proteins. They, however, found

49
that the levels of the amino acids, cysteine and tryptophan
were decreased by 25 and 8%, respectively. Moran and
coworkers (1953) reported that methionine was oxidized to the
corresponding sulfoxides and sulfones and cysteine to cysteic
acid in flour treated with chlorine dioxide. Ewart (1968)
exposed solutions of wheat proteins to chlorine dioxide and
observed the formation of a pink-yellow color due to the
oxidation of tryptophan and tyrosine. Under similar
conditions there was an increase in chlorine dioxide-induced
solubility of flour proteins. In the treatment of wool,
chlorine dioxide has been reported to cause a dissolution of
up to 40% of the proteinaceous matter leading to losses in
weight. These weight losses have been attributed to the
reactivity of amino acid components of the wool with chlorine
dioxide (Masschelein, 1979). Tyrosine, methionine, glutamate
and tryptophan were the most reactive. The reactivity of
tryptophan with chlorine dioxide was found to be greater than
that of tyrosine and usually led to the formation of a reddish
coloration (Masschelein, 1979) .
Toxicological Assessment
Evidence already presented suggests that both chlorine
and chlorine dioxide have the capacity to react with a wide
range of organic compounds under different treatment
conditions. Many organic substances in food and potable water
are capable of reacting with chlorine to produce both

50
halogenated and oxidized products. Chlorine dioxide on the
other hand produces mainly oxidized reaction products. Even
the most gentle chlorination treatments are likely to produce
chlorinated and non-chlorinated organics. Chlorination of
organic compounds has been shown to increase their lipophilic
nature and thereby increase their toxicity and/or their
ability to bioaccumulate (Kopperman et al., 1978). This
section will focus on the toxicology of chlorine and compounds
arising from the use of chlorine and chlorine dioxide
disinfection and their possible health hazards.
Overview
Chlorine has been used as the major disinfectant for
water supplies for many years and has been a major factor in
reducing the transmission and spread of numerous waterborne
diseases and infections. Concerns about the use of chlorine
for water treatment came to light when it was discovered that
THMs are produced as a result of water chlorination practices
(Bellar et al., 1974; Rook, 1974). These concerns gained
additional impetus when the National Cancer Institute reported
that chloroform, a major byproduct of chlorination, is
carcinogenic in two laboratory animals (NCI, 1976). Because
of this discovery, the United States Environmental Protection
Agency amended the National Interim Primary Drinking
Regulation which set up guidelines for the control of THM
concentrations in drinking water. A maximum contaminant level
of 0.10 mg/L was set for total THMs in finished drinking water

51
in the United States (Symons et al., 1981). In order to
comply with these guidelines, several researchers in the area
have devoted their efforts to the search for alternate
disinfectants which will produce very little or no THM in
finished drinking water (Condie, 1986; Lykins and Griese,
1986; Symons et al., 1978).
Chlorine dioxide was selected as the alternate oxidant
and disinfectant for drinking water because in most aspects
it is similar or superior to chlorine and it produced very
little or no THM (Knocke et al., 1987; Aieta and Berg, 1986;
Lykins and Griese, 1986; Lauer et al. , 1986; Aieta et al.,
1980) . Also, it has been in use for many years in several
European countries without any reported adverse effects
(Symons et al., 1978). According to Symons and coworkers, a
1977 survey showed that about 103 facilities were using or had
used chlorine dioxide in the United States. The suggested use
of chlorine dioxide was welcomed by several researchers and
numerous reviews are available regarding its action,
especially for THM control. One of the main disadvantages of
chlorine dioxide is its additional cost to the consumer which
is estimated at about three times that of chlorine. However,
a report by Lykins and Griese (1986) in Evansville, Indiana,
shows that apart from being suitable for THM control, chlorine
dioxide is also cost effective (about $1.77/resident/year).
On the other hand, it becomes clear that the toxicological
problems and possible health hazards associated with chlorine

52
dioxide and its disinfection byproducts are not immediately
known (Condie, 1986).
Unfortunately, THMs are not the only byproducts of
disinfection which pose possible health hazards in drinking
water. Other chlorinated and non-chlorinated organic
compounds, both volatile and non-volatile, have been reported
to be produced during water chlorination (Loper et al., 1985;
Coleman et al., 1983 ; Cheh et al. , 1980; Christman, 1980).
Organic compounds that have been identified in drinking water
are mostly volatile and represent only about 5-10% of the
total organic compounds in drinking water (NAS, 1977). Of the
250 or more compounds previously identified in drinking water,
about 10% have been classified as being carcinogenic and a
good number remain to be assessed for genotoxicity (Kraybill,
1978). According to the latest survey, more than 1,100
organic compounds have been identified in drinking water, most
of which are at or below the ^q/L level (Lucas, 1985).
Another group of compounds, haloacetonitriles (HANs),
has been identified in drinking water which has compounded
the problem (Daniel et al., 1986; Bierber and Trehy, 1983;
Oliver, 1983; Trehy and Bierber, 1981). Proteinaceous
material, especially amino acids, as well as fulvic and humic
substances have been implicated and confirmed precursors of
both THMs and HANs upon reaction with chlorine (Bierber and
Trehy, 1983; Oliver, 1983; Rook, 1980). A third class of
compounds which has received very little attention is the

53
relatively high molecular weight compounds which are not
easily amenable to gas chromatographic analysis. This class
of compounds is mostly non-volatile and several have been
shown to be mutagenic in short-term assays (Cheh et al.,
1980). The difficulty encountered in their isolation and
identification is that they are mostly non-volatile and
difficult to identify using current analytical techniques
(Meier et al., 1983). Some are polymerization products
produced from the condensation of several individual compounds
which makes identification more difficult even with up-to-date
instrumentation. Due to these analytical limitations, the
toxicology and possible health risks posed by this class of
compounds still remain unknown.
Toxicology of chlorine and chlorine dioxide
Chlorine forms hypochlorous acid (H0C1) which dissociates
to form hypochlorite ion (0C1‘) in solution. Under alkaline
conditions, chlorine dioxide disproportionates to form
chlorite (C102’) and chlorate (C103‘) . Most of the current
literature on the toxicology of chlorine dioxide and chlorine
deals with the undesirable byproducts arising from the
reactions of these disinfectants with organic materials in
water. As a result, very little information is available on
the toxicology of the disinfectants themselves or the
endproducts of their decomposition such as chlorite, and
hypochlorite. This section reviews the available work
concerning the toxicology of chlorine and chlorine dioxide,

54
as well as their decomposition byproducts, hypochlorite,
chlorate and chlorite.
Chlorine, hvoochlorous acid and hypochlorite
Information dealing with the toxicity of chlorine is
somewhat conflicting. Blabaum and Nichols (1956) reported
that no gross abnormalities occurred in mice fed chlorinated
drinking water for 30 or 50 days. Also, a seven generation
study in rats administered chlorine in their drinking water
did not result in any observable adverse toxic or teratogenic
effects. Other reports on the toxicity of chlorine or
chlorinated water indicate that toxicity, if any, is not due
to chlorine itself but probably due to its reaction with
precursors in the stomach. Chang et al. (1981) have reported
that administration of chlorinated water led to the
development of fatty liver in rats with a better than two-fold
increase in liver triglycerides. The acyl groups of
triacylglycerols and phospholipids were also affected. Such
effects were completely reversible when dosing was
discontinued and the animals recovered fully after 10 days.
The induction of fatty liver may be due to the formation of
chloroform with precursor substances in the body since
chloroform has been shown to give rise to this condition
(Cornish, 1980).
No toxic effects were observed in rats and guinea pigs
fed up to 400 ppm of sodium hypochlorite in milk and water
(Cunningham, 1980). At low dose levels, chlorine was actually

55
shown to act as a broad spectrum antibiotic and stimulated the
growth rates of the rats and guinea pigs. However, at very
high doses (2000 ppm), Cunningham observed significant
increases in liver weights probably due to the formation of
fatty liver described by Chang and coworkers (1981). In
earlier studies, Cunningham and Lawrence (1977) fed flour,
chlorinated with 2,000 and 10,000 ppm chlorine, to rats and
observed decreased growth rates and increased liver and kidney
weights. The authors reported similar effects in rats fed
chlorinated flour lipids and proteins (Cunningham and
Lawrence, 1978).
The tumor initiating and/or promoting activities of
chlorine have also been studied but the results are still
inconclusive. Hayatsu and coworkers (1971) observed that
sodium hypochlorite acts as a cocarcinogen in mice and
increased tumor yields when applied in conjunction with a
sub-threshold dose of 4-nitroquinoline-l-oxide. Contrary to
these observations, Pfeiffer (1978) reported that sodium
hypochlorite decreased the carcinogenic activity of
benzo(a)pyrene in mice.
Chlorine has been reported to induce chromosomal
aberrations in mammalian cells in vitro (Mickey and Holden,
1971), and sodium hypochlorite, which is commonly used in
water disinfection, has been shown to inhibit preferentially
the growth of DNA polymerase-deficient strains of E. coli
(Rosenkranz, 1973). These mutagenic events are due to the

56
reaction of chlorine with the DNA of living matter
(Rosenkranz, 1973). Sodium hypochlorite and not hypochlorous
acid (Thomas et al., 1987) has also been reported to be weakly
mutagenic for Salmonella tvphimurium in the Ames Salmonella/
microsome assay (Wlodkowski and Rosenkranz, 1975). It
resulted in a base-pair substitution mutation in Salmonella
presumably due to a reaction with purine and pyrimidine bases
which has previously been demonstrated to occur in vitro
(Hoyano et al., 1973). Meier et al. (1983) have demonstrated
that sodium hypochlorite, and not hypochlorous acid, was
capable of inducing sperm-head abnormalities in mice when fed
various chlorine compounds by gavage. From the evidence
provided, it is not clear whether the mutagenic and/or
carcinogenic effects are due to chlorine itself, or due to the
action of some reaction byproducts. It has been reported that
chloroform, di- and trichloroacetic acid as well as di- and
trichloroacetonitrile were formed in vivo in rats following
oral administration of sodium hypochlorite (Mink et al.,
1983) . Some of these byproducts may play a role in the
observed genotoxic effects and will be discussed in the latter
section.
Recent studies by Revis and coworkers (1985) have shown
that chlorine residuals similar to those found in drinking
water increased heart rate, heart fibrous tissue and plasma
cholesterol levels in rabbits and pigeons by interacting with
a diet marginal in calcium. They suggested a relationship

57
between
water
chlorination
and
hypothyroidism
which they
assumed
to be
caused
by
the
production of
chlorinated
products
In
earlier
studies
Revis et al.
(1983) also
observed signs of myocardial hypertrophy and arteriosclerosis
in rabbits and pigeons fed chlorine as sodium hypochlorite in
their drinking water. These changes were reversed when
calcium intakes in the diets were restored to normal.
Chlorine, as chloramine, has received very little
attention in terms of its toxicological potential. It has
been reported to be weakly mutagenic in bacteria (Shih and
Lederberg, 1976) but does not produce sperm-head abnormalities
in mice (Meier et al., 1985). In a very recent study, both
mono- and dichloramines and their derivatives were shown to
be mutagenic in the Ames assay (Thomas et al., 1987). The
authors showed that S. tvphimurium strain TA100 was more
sensitive to chloramine mutagenesis and that the lipophilic
dichloramines were more active than the corresponding
monochloramines. Chloramine as the synthetic disinfectant,
chloramine-T, has also been reported to cause sister chromatid
exchanges in cultured Chinese hamster ovary cells in vitro
(Weitberg, 1987). Chloramine has also been evaluated for
teratogenicity in developing rat fetuses but showed little or
no adverse effects at levels up to 100 ppm (Abdel-Rahman et
al., 1982) .

58
Chlorine dioxide, chlorate and chlorite
The toxicology of chlorine dioxide as well its inorganic
byproducts, chlorate (C103") and chlorite (C102‘) , has been
studied extensively because of their observed effects on the
hematopoietic system. All three compounds are usually
evaluated together because both chlorine dioxide and chlorate
can rapidly be converted to chlorite, and therefore, some
aspects of their toxicology are expected to be similar
(Condie, 1986). Like chlorine, considerable disagreement
exists in the literature concerning the toxicity of chlorine
dioxide and its reaction products. Chronic administration of
chlorine dioxide and chlorate to rats in their drinking water
did not produce any observed toxic effects at doses up to 10
mg/L. However, higher mortality rates were observed in rats
administered 100 mg/L for 2 years (Haag, 1949).
Chlorite has been shown to be a potent stressor of blood
and produces methemoglobinemia and hemolytic anemia both in
vivo and in vitro (Hefferman et al., 1979a; 1979b). The dose
required to produce hemolytic anemia is much lower than that
required for methemoglobinemia. It has also been reported to
cause hemolytic anemia, increase osmotic fragility and
glucose-6-phosphate dehydrogenase activity in erythrocytes,
and reduce growth and conception rates in mice (Moore and
Calabrese, 1980a; 1980b; Moore et al., 1980). Further studies
have established that all three compounds produce the same
hemolytic effects in test animals as well as decrease

59
glutathione levels in rats (Abdel-Rahraan et al., 1984;
Abdel-Rahman et al., 1979; Couri and Abdel-Rahman, 1979).
Chlorine dioxide induced reduction in glutathione levels
tended to disappear with continued exposure (Couri and
Abdel-Rahman, 1979). The mechanism of the hemolytic activity
of chlorine dioxide and its metabolites has been attributed
to the production of hydrogen peroxide which, like other
strong oxidants, produces this effect (Bull, 1983).
Chlorite and chlorate, but not chlorine dioxide have been
reported to produce hematological effects in nonhuman primates
(Bercz et al., 1982). Chlorine dioxide, on the other hand,
caused a decrease in serum thyroxin levels in monkeys. The
hypothyroid effect was unique to chlorine dioxide since its
endproducts, chlorite and chlorate, did not show this effect
even at higher doses (Bercz et al., 1982). Since chlorine
dioxide is a very reactive oxidant, it is not known whether
the decrease in thyroxin levels is due solely to this
disinfectant or due to some primary oxidation products formed
with precursor molecules in the gastrointestinal tract. Other
studies showed no significant decreases in serum thyroxin
levels in adult rats fed up to 100 mg/L chlorine dioxide
(Condie, 1986). However, administration of similar doses to
pregnant and lactating females resulted in decreased serum
thyroxin concentrations in rat pups (Condie and Bercz, 1985;
Orme et al., 1985).

60
Reproductive and teratogenic studies have also been
performed in laboratory animals. Exposure of female mice to
chlorine dioxide in their drinking water from breeding to
weaning resulted in reduced growth rates and body weights
(Moore et al., 1980), and at much higher concentrations,
increased stillbirths and fetal reabsorptions (Couri et al.,
1982). Contrary to these findings, Suh and coworkers (1983)
did not observe any maternal toxicity or gross changes in
fetal weight, litter size or skeletal anomalies in the
development of rats fed chlorine dioxide, chlorite and
chlorate in their drinking water. Further studies did not
show any measurable adverse reproductive or teratogenic
effects in rats fed sodium chlorite in drinking water (Carlton
and Smith, 1985). Carlton and Smith (1985) showed that
chronic exposure of rats to 100 mg/L sodium chlorite increased
the number of abnormal sperms and to some extent, impaired
sperm motility. This finding is in contrast to that of Meier
and coworkers (1985) who did not detect any sperm-head
abnormalities in mice dosed up to 400 mg/L chlorine dioxide,
chlorate and chlorite.
The effects of chlorine dioxide on the neurobehavioral
development of rats were investigated by Taylor and Pfohl
(1985). They observed that administration of chlorine dioxide
both postnatal and prenatally resulted in behavioral defects
and depressed brain growth. These effects were consistent
with depressed thyroid function. The authors also observed

61
delayed eye opening, decreased body and brain weights and
activities, and delayed locomotor activity. Chlorine dioxide,
and not chlorite, has been implicated as a hyper-
cholesterolemic agent which increased serum cholesterol levels
by interacting with a diet marginal in calcium (Revis et al.,
1985) . No definitive conclusions have been made on the
genotoxicity of chlorine dioxide and its metabolites. Suh et
al. (1984) reported that chlorine dioxide depressed DNA
synthesis in the testis and intestinal epithelium of rats
dosed 100 mg/L chlorine dioxide in their drinking water.
Meier et al. (1985) tested the mutagenic activity of chlorine
dioxide, chlorate and chlorite in mice using chromosomal
aberrations, micronucleus and sperm-head abnormalities assays
as endpoints. No mutagenic activity was detected in any of
the disinfectants 5 days after exposure. The tumor initiating
and promoting activities of chlorate and chlorite have also
been investigated. Both chemicals did not demonstrate any
significant carcinogenic activity in Senear mice (Kurokawa et
al., 1985? 1984).
With regard to human studies, oral administration of
chlorine dioxide, chlorate and chlorite to healthy adult male
volunteers did not produce any detrimental clinical effects
(Lubbers et al., 1985). The authors employed an exposure time
of 12 weeks with the volunteers receiving a total of 5 mg/L
chlorine dioxide or its endproducts. Again, no biochemical
or physiological effects were observed in the volunteers.

62
Caution must be taken in interpreting these findings since a
lack of effect does not necessarily imply safety. It is not
known whether long-term low-dose exposure to any of these
chemicals will induce any severe adverse physiological
effects.
Mutagenesis and Carcinogenesis
Different classes of compounds have been identified in
drinking water, some of which may be produced from
chlorination reactions. A 1976 USEPA survey showed that of
289 compounds identified in drinking water, approximately 38%
were halogenated. Another survey of five major cities in the
United States showed that approximately 50% of the volatile
hydrophobic compounds in drinking water were halogenated
(USEPA, 1975). The number could be considerably higher if
proper analytical methods for detection and quantification
were available. Also, the source and distribution of these
chemicals differ. This section will briefly review the
mutagenic and carcinogenic properties of those compounds that
are formed during water chlorination, such as THMs and HANs,
and those of precursor substances such as amino acids and
humic substances.
Drinking water
The carcinogenic properties of drinking water were first
reported by Marx (1974). Since then numerous studies have
been conducted on the mutagenic and carcinogenic properties
of drinking water in different municipalities in the United

63
States and elsewhere and, in all cases, researchers have shown
that mutagens are produced as a result of water chlorination.
Drinking water in the United States and Canada (Basu et al.,
1985; Nestmann et al., 1983; Cheh et al., 1980; Loper, 1980),
South Africa (Grabow et al., 1980), the Netherlands (Kool et
al., 1985), United Kingdom (Horth et al., 1987; Fielding and
Horth, 1986), Finland (Kronberg et al., 1988), Israel
(Guttman-Bass et al., 1987) and many other countries have been
tested and reported to be mutagenic in several short-term
assays. In most of the studies, replacement of chlorine with
chlorine dioxide leads to a reduction in the observed
mutagenic activities (Guttman-Bass et al., 1987). According
to the findings of Kool et al. (1985), chlorine treatment of
drinking water generally increases the mutagenic activity in
S. typhimurium TA98 and TA100. Also chlorine dioxide is
capable of increasing the mutagenic activity in certain cases
with the overall increase being less than that of chlorine.
The main drawback to these findings is that mutagenicity
is usually detected from a mixture of compounds of different
classes and origin, and even though compounds such as
chloroform, bromoform and tetrachloroethylene are known
carcinogens, their overall contribution to the observed
genotoxicity is not usually known. Being highly volatile,
some of these chemicals may be lost during sample
concentration procedures prior to genotoxicity evaluation.
The third problem is that some of these compounds are poorly

64
characterized non-volatiles which cannot be easily isolated
and identified, therefore, their overall contribution to the
carcinogenic and mutagenic effects remain unknown. None of
the studies in the literature has accounted for 100% of the
mutagenic activity observed in drinking water extracts.
Kronberg et al. (1988) have recently identified a very strong
mutagen,3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone
(MX) and its isomer, (E)-2-chloro-3-(dichloromethyl)-4-
oxobutenoic acid (EMX) in acidified chlorinated extracts of
humic acid and drinking water. According to the authors, MX
accounted for 50-100% of the mutagenic activity of extracts
of chlorinated humic water and for 20-50% of the activity in
drinking water extracts while EMX accounted for only about 2%
of the total mutagenic activities using the Ames Salmonella
assay. MX has also been detected in drinking water from three
locations in the U.S.A. and in the chlorination of humic acid
(Meier et al., 1987). According to the authors, MX appears
to account for a significant proportion of the mutagenic
activity observed in these samples.
Presented in Table 1 is a of some of the compounds
identified in drinking water which includes known or suspected
carcinogens. Volatile compounds have been defined as those
that are purgeable from drinking water at room temperature
with an inert gas such as helium (Tardiff et al., 1978).
These compounds represent 5-10% of the total organics in
drinking water and the remaining 90-95% have not been fully

65
Table 1. Some halogenated compounds formed in drinking water
as a result of water chlorination3
Chloroform6
Trichloroacetic acid
Bromodichloromethane6
Trichloroacetaldehyde
Chiorodibromomethane6
Chlorophenols
Bromoform6
A1pha-chioroketones
Dichloromethane
Chlorobenzenes
H H
Bis (2-chloroethyl) ether Chlorinated acetonitriles
Trichloroethylene6
Chlorinated aromatic acids
Tetrachloroethylene6
Chlorinated purines
1,1,l-Trichloroethane
Chlorinated pyrimidines
aAdapted from Kraybill
(1978)
'Known or suspected carcinogens

66
identified, or are unknown (NAS, 1980). Not included are
those high molecular condensation and/or polymerization
products which are not easily amenable to gas chromatographic
analyses even with sample derivatization.
Trihalomethanes and haloacetonitriles
THM were the first class of compounds which aroused
interest in chlorination byproduct formation (Rook, 1974;
Bellar et al., 1974). Most of the purgeable organics are
formed in drinking water at a level of 1 ppb or less.
However, chloroform is usually present in both treated and
untreated water at much higher concentrations of up to 100
ppb on the average (Tardiff et al., 1978; Bellar et al.,
1974). Some of the most frequently encountered chlorinated
compounds in groundwater include trichloroethylene, vinyl
chloride, tetrachloroethylene, 1,1-dichloroethylene, carbon
tetrachloride, 1,1,1-trichloroethane and 1,1-dichlorobenzene
(Cotruvo and Vogt, 1985). Chloroform, the major THM, has been
shown to be carcinogenic and to increase the incidence of
hepatocellular carcinomas and kidney epithelial tumors when
administered to rats and mice (NCI, 1976). However, recent
studies indicate otherwise (Pereira et al., 1985). In 1983,
Pereira showed that THM, including chloroform, possess very
little or no tumor initiating activity and that their main
action in tumorigenesis is promotion. Pereira et al. showed
that chloroform, apart from low level DNA binding, did not
possess any tumor initiating property. In a fairly recent

67
study, Pereira and coworkers (1985) administered 1,800 ppm
chloroform to Swiss mice, pretreated with ethylnitrosourea,
and reported that chloroform inhibited hepatocarcinomas in the
mice. According to the authors, administration of chloroform
in the drinking water of Swiss mice inhibited both spontaneous
and induced hepatocarcinogenesis caused by ethylnitrosourea.
These results are in sharp contrast to the NCI study in which
chloroform was shown to be a hepatocarcinogen. In the NCI
study, chloroform was administered in corn oil and Pereira
and coworkers point out the possibility of a synergistic
interaction between the vehicle and chloroform leading to the
previously observed hepatocarcinogenicity.
Chloroform has been reported to be non-mutagenic in
bacteria assays including Salmonella and E. coli (Simmon et
al., 1977; Uehleke et al., 1976). Other THMs such as
bromoform, bromodichloromethane and dibromochloromethane were
only weakly mutagenic in Salmonella without metabolic
activation (Simmon et al., 1977).
The second major class of volatile organics, HANs, have
received considerable attention since they were discovered
(Oliver, 1983; Bierber and Trehy, 1983). Bierber and Trehy
(1983) reported that HANs easily escape detection because they
undergo gradual hydrolysis to form nonvolatile products.
Dichloroacetonitrile, the major HAN, is usually detected in
drinking water at about 10% that of the average THM
concentration (Oliver, 1983). Haloacetonitriles (HAN) have

68
been reported to pose some possible health problems to humans
because of their potential mutagenic and carcinogenic
properties in test animals (Meier et al., 1983). They have
been detected in the stomach contents of rats dosed with
aqueous chlorine solutions and the possibility exists for the
same effects to be observed in humans (Mink et al., 1983).
Bull and Robinson (1985) tested the mutagenic activity of the
five common HAN, chloroacetonitrile, dichloroacetonitrile,
trichloroacetonitrile, bromochloroacetonitrile, and
dibromoacetonitrile in the Ames Salmonella/microsome assay and
found that dichloroacetonitrile and bromochloroacetonitrile
were direct-acting mutagens which produced base-pair
substitution mutations in Salmonella. All five compounds
induced significant elevations in sister chromatid exchange
(SCE) frequencies in Chinese hamster ovary (CHO) cells. SCE
frequencies increased with increasing chlorine substitution
and was further enhanced when bromine was substituted for
chlorine. Three of the compounds were also capable of
producing tumors in Senear mice. HAN have been shown to bind
covalently to liver and kidney DNA and cause DNA strand
breaks, which indicates potential carcinogenic ability
(Pereira et al., 1985). Trichloroacetonitrile was found to
be the most potent DNA strand breaker while the bromo-
derivatives were intermediate. Pereira and coworkers have
proposed a scheme for HAN metabolism which involves formation
of phosgene, a highly reactive electrophile, and cyanide.

69
The above findings were further confirmed by Daniel et
al. (1986) who reported that HANs were cytotoxic in human
cultured lymphoblast (CCRF-CEM) cells. They produced 13-60%
cell killing at a dose of 50 ¿mol and caused major DNA strand
breaks. It should be noted that in all of these studies,
chlorine dioxide has not been mentioned due to the fact that
its use in water treatment does not lead to THM formation.
Further studies are needed to determine the stability of HANs
in aqueous media and the possible health hazards associated
with their long-term low-dose exposure to humans.
Precursor substances
The final part of this review will focus on those
precursor substances which react with chlorine or chlorine
dioxide to form genotoxicants. Proteinaceous material and
humic and fulvic substances are known precursors of
haloacetonitrile and trihalomethane formation in natural
waters upon reaction with chlorine. Unfortunately, these
compounds are not the only ones of concern in drinking water
since other chlorinated and non-chlorinated byproducts are
formed as well. Even though chlorine dioxide produces very
little or no THM in water, it does not remove them from water.
Also, some of the oxidation byproducts of chlorine dioxide
reactions in water may also be genotoxic.
Amino acids, peptides and proteins. The genetic effects of
amino acids after chlorination were first reported by Sussmuth
(1982) who demonstrated that harmless nutrients such as amino

70
acids and yeast and meat extracts could be converted into
carcinogens and mutagens by chlorination. In this experiment,
80 mL of chlorine gas were bubbled through 250 mg of yeast
extract, meat extract or amino acids for 4 minutes and the
reaction products evaluated for genotoxic activity. Due to
the high chlorine dose, Sussmuth did not find the need to
concentrate the reaction products. Methionine was found to
be the most potent precursor of genotoxic activity both in the
Ames Salmonella assay and the rec assay in Bacillus subtilis.
Tyrosine, phenylalanine, cysteine and glycine were also
genotoxic. In the mutagenicity assay, all the above compounds
were direct-acting mutagens which did not reguire metabolic
activation. None of the reaction products were identified in
this study.
In another study, Horth et al. (1987) evaluated the
mutagenic activity of several amino acids in Salmonella after
reaction with sodium hypochlorite. With the exception of
glycine, mutagenic activity was detected in all the amino
acids treated with chlorine. Methionine, phenylalanine,
tyrosine and a group of heterocyclics were found to be the
most potent precursors of mutagenic activity. Of the
heterocyclics, tryptophan and proline were the most active.
The authors identified several compounds in their mutagenic
extracts. Except for dichloroacetonitrile which is a known
carcinogen, none of the other compounds identified possessed
mutagenic activity when tested separately. It was concluded

71
that the level of dichloroacetonitrile was too low and
unlikely to account for any significant portion of the
mutagenicity. In follow-up studies Horth and coworkers (1989)
tentatively identified several other compounds from
chlorination of tyrosine and phenylalanine. Using authentic
standards, the authors showed that dichloroacetonitrile,
produced from both amino acids, and benzylchloride and
2-phenyl-2,2-dichloroethenal, produced from phenylalanine,
were all mutagenic to S. typhimurium strain TA100.
Dichloroacetonitrile has also been shown to be produced from
the chlorination of aspartic acid, tyrosine and tryptophan
(Trehy et al., 1986). The overall contribution of the
unidentified compounds and those for which no authentic
standards were available still remain unknown.
In similar studies Rapson and coworkers (1985) tested
the mutagenicity of compounds produced by reaction of aqueous
chlorine and chlorine dioxide with tyrosine, at increasing
chlorine to tyrosine ratios. Tyrosine produced mutagenic
reaction products with the mutagenicity increasing with
chlorine dose. Mutagenicity peaked at 4 equivalents of
chlorine per mole of tyrosine. Substitution of chlorine with
chlorine dioxide led to a linear decline in mutagenic activity
from 2,100 revertants over background to 0 with 100% chlorine
dioxide. Similar results were obtained by Tan et al. (1987b)
who showed that replacement of chlorine with chlorine dioxide
on a molar basis decreased the mutagenic activity in

72
chlorinated tryptophan concentrates. However, in the case of
two dipeptides, L-tryptophylglycine and L-glycyl-L-tryptophan,
reaction with chlorine dioxide led to the formation of
mutagens while aqueous chlorine did not (Tan, 1986) . In
contrast to the findings of Sussmuth (1982) and Horth et al.
(1987), Tan et al. (1987b) did not detect any mutagenic
activity after reaction of aqueous chlorine and methionine.
Chlorination of purines and pyrimidines as well as
nucleosides and nucleotides did not result in any mutagen
formation (Horth et al., 1987). It is evident that many
difficulties exist in the concentration, fractionation and
evaluation of individual components in drinking water or model
systems. In a majority of tests, a mixture of compounds and
not individual components are used due to analytical
difficulties encountered sample separation. Unless proper
separation and analytical methods are developed, it will be
difficult to pinpoint the individual chemical or group of
chemicals responsible for mutagenic activity in chlorination
reaction mixtures.
Fulvic and humic substances. Fulvic and humic acids are
naturally occurring metabolic endproducts of decaying
vegetation which account for the bulk of organic matter
present in surface waters. Chlorination of water containing
these substances has been reported to result in the formation
of THMs and other non-chlorinated, high molecular weight,
poorly characterized organics (Coleman et al., 1983; Rook,

73
1977) , some of which are mutagens and carcinogens (Kronberg
et al. , 1988; Meier et al., 1987, 1986 and 1983).
Chlorination of kraft wood pulp, catechol, lignin and
humic and fulvic acids have all been shown to give rise to
mutagen formation in the Ames Salmonella/microsome assay
(Kronberg et al., 1988; Meier et al., 1987; Holmbom et al.,
1984; Kringstad et al., 1983; Meier et al., 1983; Nazar and
Rapson, 1982; Nazar et al., 1981). The pH of the reaction is
important in mutagen formation especially when Amberlite XAD
resins are used to adsorb or concentrate the reaction
mixtures. According to Nazar and Rapson (1982), at elevated
pH of 7 or higher, mutagenic activity decays due to the
cleavage of organically bound chlorine by hydroxide ions.
These findings have been confirmed by Meier et al. (1983) who
observed a decrease in mutagenic activity with increasing pH
due in part to the alkali lability of the compounds.
Mutagenic activity of chlorinated model humic acids is similar
to that of drinking water which implies that these substances
account for a substantial portion of the mutagenic activity
of drinking water (Kronberg et al., 1988; Meier et al., 1987;
Coleman et al., 1984). Also most of the mutagenic activity
is due to non-volatile compounds since most of the volatile
organics are lost during sample concentration (Meier et al.,
1983). Coleman et al. (1984) identified more than 25
compounds from ether extracts of chlorinated humic acids and
confirmed their individual mutagenic activities using

74
authentic standard compounds. Kronberg and colleagues have
also identified and characterized a strong mutagen, MX, from
acidified extracts of chlorinated humic acid using HPLC
fractionation techniques. MX accounted for 50-100% of the
observed mutagenic activity while its isomer, EMX, accounted
for very little of the observed activity. In a separate
study, Meier et al. (1986) identified several compounds from
ether extracts of chlorinated humic acids, some of which are
known mutagens. Analysis of these extracts indicated that
they accounted for only 7% of the total activity in the
original solutions. They also identified MX and EMX which had
previously been identified (Holmbom et al., 1984). Further
studies by Meier et al. (1987) and Hemming et al. (1986)
showed that MX accounted for between 15-34% and 3-19%,
respectively, of the mutagenic activity in XAD concentrates
of drinking water. Both research groups observed that sample
acidification prior to XAD adsorption was essential for the
effective recovery of MX from reaction drinking water or
chlorination reaction mixtures.
In summary, evidence has been provided to show that
genotoxicants are produced during chlorination of water which
contains proteinaceous material and fulvic and humic
substances. A comparison of the mutagenicities of chlorinated
and non-chlorinated surface waters has always shown that
mutagens are present in the chlorinated samples. In any
environmental impact assessment study, those compounds that

75
are not easily identifiable should be taken into account in
order to ensure that they are not being ignored.

CHAPTER III
MATERIALS AND METHODS
Chlorine Demand-free Water
Chlorine demand-free water was prepared following a
modification of the procedure of Ghanbari et al. (1982a).
Distilled water was passed through two successive Barnstead
deionizing units (Barnstead, Division of Sybron Corporation,
Boston, MA) and then through a glass column containing Porapak
Q (Supelco, Inc., Bellefonte, PA). The water was analyzed by
iodometric titration (APHA, 1985) to determine the presence
of any available chlorine. Water used for the preparation of
all the reagents and substrates was prepared this way.
Generation of Aqueous Chlorine
Commercial chlorine is contaminated with a variety of
organochlorine compounds including chloroform, phosgene,
carbon tetrachloride, methylene chloride, etc. (Christman,
1983) . To ensure that no such contaminants were present in
the chlorine sample used in this study, a modification of the
methods of Ghanbari et al. (1983) was used for the generation
of agueous chlorine. Chlorine gas was prepared by dropwise
addition of 3 N HC1 to KMn04 (Fisher Scientific, Fair Lawn,
NJ) in a closed system under vacuum. The liberated gas was
76

77
trapped in ice-cold chlorine demand-free water or 0.1 M sodium
phosphate buffer, pH 7 as desired. Under these generation
conditions, the gas was efficiently trapped mainly as
hypochlorous acid. This was detected by a drop in the pH of
the solution from 7.0 to 2.5-3 due to the liberation of
hydrogen ions. The purity and available chlorine
concentrations of the stock solutions were determined by
iodometric titration (APHA, 1985). Aqueous chlorine solutions
were diluted when necessary and used immediately after
preparation.
Generation of Aqueous Chlorine Dioxide
Stock solutions of aqueous chlorine dioxide were prepared
by slow addition of 2 N H2S04 to 6 g sodium chlorite (Eastman
Kodak Co., Rochester, NY) in 6 mL water in a closed system
(APHA, 1985). The chlorine dioxide gas generated from the
reaction was passed through a sodium chlorite clean-up column
to remove any contaminants such as chlorine. The gas was then
collected as a greenish-yellow solution in ice-cold chlorine
demand-free water or 0.1 M sodium phosphate buffer as desired.
As before, the concentrations of chlorine dioxide solutions
were determined iodometrically. To prevent losses due to
volatilization and/or breakdown, chlorine dioxide solutions
were always prepared fresh, diluted when necessary, and used
immediately after preparation. All chlorine and chlorine

78
dioxide stock solutions were prepared under yellow lights to
prevent photodecomposition.
Iodometric Titration
Iodometric titration was used to determine available
chlorine and chlorine dioxide concentrations in all the stock
solutions as well as chlorine residuals remaining after the
reactions. The method involves liberation of iodine from
potassium iodide (KI) by chlorine. The liberated iodine is
then titrated with sodium thiosulfate (Na2S203) to a starch end
point (APHA 1985) . Excess KI acidified with acetic acid to
pH 3-4 was mixed with aliguots of the stock chlorine or
chlorine dioxide solutions in glass-stoppered flasks at room
temperature. In the case of chlorine dioxide solutions, the
chlorine dioxide-KI solutions were kept in the dark for about
5 minutes prior to titration. The mixtures were then titrated
under yellow lights with 0.01 N Na2S203 solution prepared by
diluting from standardized stock solutions, until the yellow
color of the liberated iodine was almost clear. A few drops
of a 0.5% starch indicator solution were added and titration
was continued to a colorless end point. A blank titration
using only chlorine demand-free water was also performed to
correct for interferences from oxidizing and reducing
substances and to correct for the concentration of iodine
bound starch at the end point (APHA, 1985).

79
The total available chlorine or chlorine dioxide was
calculated as follows:
mg Cl or C102 as Cl2/L (ppm) = (A ± B) x N x 35450
mL sample
where: A = Volume of Na2S203 used for the sample,
B = Volume of Na2S203 used for the blank,
N = Normality of the Na2S203 used.
Reactions of Aqueous Chlorine or Chlorine Dioxide with
L-Trvptophan
Preliminary reactions of aqueous chlorine
In preliminary experiments, 0.1 M L-tryptophan (Sigma
Chemical Co., St. Louis, MO) solutions were prepared in 0.1
M sodium phosphate buffer at pH 7.0 with slight warming to
ensure that all the amino acid was dissolved. Two molar
ratios of aqueous chlorine to amino acid, 1:1 and 3:1, and a
reaction time of 24 hours was used to ensure that the reaction
was complete. This study used aqueous chlorine alone with pH
adjustment of the reaction mixture after 24 h to obtain
acidic, neutral and basic fractions. Each fraction was
extracted separately first with hexane, then with methylene
chloride and lastly with ethyl ether.
Reactions of aqueous chlorine and chlorine dioxide
In the next series of experiments, three molar ratios of
L-tryptophan to aqueous chlorine or chlorine dioxide, 1:1, 1:3
and 1:7 (M:M), were chosen. Tryptophan solutions were

80
prepared in 0.1 M sodium phosphate buffer, pH 7.0, as before
and aqueous chlorine and chlorine dioxide solutions were
prepared in chlorine demand-free water. For the 1:1 and 1:3
reactions, 14 mM tryptophan solutions (approximately 2.86 g
tryptophan in 1 L sodium phosphate buffer) were reacted with
14 and 42 mM chlorinating agents (total volume 1 L),
respectively. The 1:7 reactions were carried out using a 10
mM amino acid solution (2.04 g tryptophan in 1 L sodium
phosphate buffer) and a 70 mM chlorine or chlorine dioxide
(total volume 1 L) . The amino acid concentration used for the
latter reactions were limited by the amount of chlorinating
agent generated by the above procedures. All the reactions
were carried out using equal volumes of reagent and substrate
(2 L final volume), therefore? the final concentrations of
chlorinating agents and amino acid were halved. Reactions
were carried out in the dark in glass jars at ambient
temperature for 24 hours with continuous mixing on a Corning
stirrer. Procedural blanks containing only amino acid,
aqueous chlorine or chlorine dioxide in sodium phosphate
buffer were used as controls.
After reaction, the samples were filtered using a 0.45
/xm membrane filter (Gelman Sciences, Inc., Ann Arbor, MI) to
remove any precipitate present. Aliquots of the reaction
mixture were titrated iodometrically to determine if any
available chlorine remained. The samples were then split in
half. The pH of one-half was readjusted to 2.5 with 85%

81
phosphoric acid and saved for resin adsorption, while the
other fraction was kept at neutral pH for liquid-liquid
extraction using ethyl ether.
Chlorine Consumption
Chlorine concentrations before and after chlorination
were measured iodometrically (APHA, 1985). Any excess
chlorine or chlorine dioxide remaining after the 24-hour
reaction was quenched using sodium sulfite (Fisher Scientific,
Fair Lawn, NJ) in order to avoid contamination with elemental
sulfur produced from dechlorination with sodium thiosulfate
(Trehy et al., 1986).
Concentration Techniques
Different techniques, mainly liquid-liquid extraction,
Amberlite XAD adsorption and column chromatography were used
to concentrate the reaction mixtures for mutagenicity and
other assays.
Liquid-liquid extraction
Preliminary studies. In preliminary liquid-liquid extraction
studies, the active ingredients in the aqueous chlorination
reaction mixtures were extracted using organic solvents of
different polarity. Ethyl ether and hexane (Fisher
Scientific) were reagent A.C.S. and methylene chloride (Fisher
Scientific) was HPLC grade. The reaction mixtures containing
aqueous chlorine and tryptophan at neutral pH were extracted

82
three times by mixing vigorously on a stirrer with equal
volumes of hexane, followed by methylene chloride and then
ethyl ether. Each extraction took approximately 2 hours and
represented the neutral fraction. After these extractions,
the aqueous mixture was divided into two portions. The pH of
one fraction was adjusted to 1.2 using concentrated HCl and
that of the other half was changed to pH 11.7 using 10 N
sodium hydroxide. These fractions were each extracted with
the three organic solvents as before to obtain acidic and
alkaline fractions, respectively, as shown in Fig. 5.
In the preliminary studies, high mutagenic activity was
detected in the ethyl ether extracts of the reaction products.
Therefore, in the latter part of the study when three ratios
of aqueous chlorine and chlorine dioxide were compared, only
ethyl ether was used for the liquid-liquid extractions.
Mixtures containing different molar ratios of aqueous chlorine
or chlorine dioxide with tryptophan were extracted vigorously
with mixing on a stirrer with the aid of a magnetic stirrer
for approximately 4 hours, and then separated with a
separatory funnel. The aqueous phase was discarded and the
ethyl ether extract was dried by rotary evaporation and saved
for further analysis.
Evaluation of product distribution in the ethyl ether
extracts. A solution containing radiolabeled 14C-tryptophan
of known activity was prepared as described below. The
reaction products were extracted with ethyl ether for 2 hours.

83
0.1 M Aqueous chlorine + 0.1 M tryptophan
in 0.1 M sodium phosphate buffer (pH 7.0)
25°C, 24 h
N/
Filter
\T
Precipitate
\1/
Aqueous phase
\K
Extract 3x with
-hexane
-methylene chloride
-ethyl ether
Aqueous phase
V
V
Organic phase
Adjust to pH 1.2 Adjust to pH 11.7
\i/ \y
Extract 3x with hexane, methylene chloride
and ethyl ether
\K
Concentrate by rotary evaporation
in vacuo
\1/
Assay for mutagenic activity
Figure 5. Schematic presentation of sample preparation
for mutagenicity assessment in the preliminary
studies

84
The ethyl ether layer was separated from the aqueous layer and
partially dried to a known volume. The percentage
distribution of radioactivity in the aqueous solution and
ether extracts was measured by dissolving aliquots in 15 mL
Scintiverse II cocktail and counted on a Beckman Instruments
liquid scintillation spectrometer as described below.
Amberlite XAD polymeric resin adsorption
Evaluation of Amberlite XAD procedure for concentrating the
reaction products. Amberlite XAD-2, a nonpolar polystyrene
adsorbent and XAD-8, an acrylic ester of intermediate
polarity, were obtained from Rohm and Haas (Philadelphia, PA) .
The resins were prepared prior to use by continuous soxhlet
extraction with acetone for 5 hours followed by further
washing with methanol for an additional 5 hours. The clean
resins were stored separately in methanol until needed. Prior
to use, the resins were slurry packed in methanol in a glass
column, 52 cm by 1.9 cm i.d. to form a bed height of 30 cm.
The resins were then rinsed with 3-5 bed volumes of distilled
water to ensure that all the methanol was removed.
A 7 mM tryptophan solution in 0.1 M sodium phosphate
buffer (pH 7.0) was prepared by dissolving 0.5 g of the amino
acid in 350 mL buffer. Radiolabeled L-tryptophan (side chain-
3-14C) with a specific activity of 53.76 mCi/mmole was obtained
from New England Nuclear, Inc. (Boston, MA) . Radiolabeled
tryptophan (0.1 mCi in 0.1 mL phosphate buffer) was mixed with

85
the tryptophan solution and the total radioactivity of the
solution was measured. Equimolar concentrations of either
aqueous chlorine or chlorine dioxide were added to separate
reaction flasks and allowed to react for 4 hours in the dark
at room temperature. The reaction mixtures were filtered
separately using a 0.45 /j,m membrane filter and the pH and
radioactivity of the solutions were measured. The solutions
were then passed through a glass column (52 cm x 1.9 cm i.d.)
containing either XAD-2 alone or in combination with XAD-8
(1:1 mixture) at a bed height of 30 cm.
Trial experiments were conducted to determine the best
Amberlite XAD concentration technique for the reaction
products. In one experiment, XAD-8 was placed on top of XAD-2
while in another the order was reversed. In another trial,
only XAD-2 was used. Also, the importance of sample pH on the
recovery of the reaction products was evaluated by adjusting
sample pH. The pH of the reaction products in one experiment
was adjusted to 2.5 with 85% phosphoric acid while in another,
it was maintained at 7.0. All the reaction products were
passed through the various resin columns at a flow rate of
approximately 40 mL/minute. The first pass effluent was
poured on the column. The adsorbed substances were eluted
successively with 200 mL each of methanol, ethyl ether and
acetone. The combined organic solvents were removed by rotary
evaporation under vacuum and the residues were diluted to 10
mL with chlorine demand-free water. Radioactivity of the

86
aqueous filtrates and concentrates were measured by counting
0.1 mL aliquots in 15 mL Scintiverse II cocktail (Fisher
Scientific, Fair Lawn, NJ) using a Beckman Instruments
spectrometer Model LS 2800 (Beckman Instruments, Inc.,
Fullerton, CA) with automatic external standardization.
Distribution of radioactivity in the filtrates and eluates
was used to measure the recovery of the reaction products.
Similar sample preparation was used to determine product
distribution in the liquid-liquid extractions.
Due to the high mutagenic activity in the Amberlite
XAD-2/8 acetone eluates of the 7:1 (aqueous chlorine or
chlorine dioxide:tryptophan) ratio, radioactivity distribution
in these eluates were also determined.
Sample concentration for mutagenicity assessment usina
Amberlite XAD resins. Based on the results from the
preliminary studies, a resin column with Amberlite XAD-8
placed on top of XAD-2 (1:1 mixture) was used for
concentrating the reaction products. The pH of the aqueous
mixtures containing the reaction products of either chlorine
or chlorine dioxide with tryptophan was adjusted to 2.5 with
85% phosphoric acid and passed through the resins at a flow
rate of approximately 40 mL/minute. This procedure was
repeated using the first-pass effluent. Control samples
containing aqueous chlorine, chlorine dioxide or tryptophan
alone in sodium phosphate buffer were dechlorinated with
sodium sulfite and also concentrated by resin adsorption.

87
After most of the aqueous fraction had passed through the
column and the water level was barely on top of the resin,
acetone was added and allowed to flow freely through the
column until it reached the bottom. Flow was interrupted and
the acetone allowed to equilibrate with the resins for 10-15
minutes. A total of 300 mL of acetone was used to elute most
of the adsorbed substances from the column. Any color or
substances remaining on the column was further eluted
successively with 300 mL methanol followed by 300 mL ethyl
ether. All the eluates were concentrated separately by rotary
evaporation (Fig. 6) . The dry samples were weighed, dissolved
in and diluted with spectrophotometric grade dimethyl
sulfoxide (DMSO) and aliquots used for the mutagenicity
studies.
Rotary evaporation
The rotary evaporator used was a Buchi Rotavapor R 110
(Brinkmann Instruments, Inc., Westbury, NY). The aqueous
reaction mixtures containing tryptophan and chlorine as well
as all the organic solvents obtained from the liquid-liquid
extractions and Amberlite XAD adsorptions were concentrated
by rotary evaporation in vacuo at 45°C.
Concentrated samples were quantitatively transferred into
preweighed teflon-capped vials and the remaining traces of
organic solvents were removed under a gentle stream of
nitrogen gas. Samples were then weighed, dissolved in and
diluted with spectrophotometric grade DMSO and used for the

88
Tryptophan solution in sodium phosphate buffer
(pH 7.0)
+
Aqueous chlorine or chlorine dioxide
chlorine to tryptophan ratio (1:1, 3:1 or 7:1)
25°C, 24 h
Dechlorinate with sodium sulfite
V
Filtration
v
Adjust to pH 2.5
V
Amberlite XAD-2/8 adsorption
V
V
Extract with ether
Elute with-acetone -
-methanol
-ether
V
V
Concentrate separately by rotary evaporation
V
Assay for mutagenic activity
Figure 6. Schematic presentation of sample preparation for
mutagenicity assessment in the reactions of
aqueous chlorine or chlorine dioxide with
tryptophan

89
Ames mutagenicity assay. Those XAD samples containing water
and the agueous concentrates were diluted in chlorine
demand-free water, aseptically filtered and aliquots used for
the mutagenicity assay.
Fractionation of Reaction Product Extracts
Highly mutagenic liquid-liquid extracts as well as
Amberlite XAD resin concentrates were further fractionated
using thin layer chromatography (TLC).
Thin layer chromatography
Uniplate silica gel G (Analtech, Inc., Newark, DE) and
Fisher brand Redi/Pit Sil gel G (Fisher Scientific), both 20
x 20 cm, were used for TLC separation. Prior to use, the TLC
plates were heat-activated for 1 hour at 150°C and allowed to
cool in a Brinkman Instruments (Westbury, NY) relative
humidity holding chamber before use. The thin Fisher brand
qualitative plates were used for preparative sample
fractionation and solvent evaluation to determine the best
solvent combinations and ratios for resolving the extracts
into single bands. A solvent system containing hexane, ethyl
ether and/or methanol acidified with acetic acid was found to
be suitable for developing the plates.
The thick Analtech silica gel plates were used for sample
preparation prior to genotoxicity evaluation. Concentrated
samples, approximately 1 mL, were applied as a thin band
across the length of the plate. The aqueous chlorine-treated

90
extracts were developed using hexane:ethyl ether:acetic acid
(50:50:2, v/v/v) while chlorine dioxide-treated samples were
developed with hexane:ethyl ether: methanol:acetic acid
(63:30:7:2, v/v/v/v). In all experiments, a total of about
3 mL of sample was fractionated this way for the mutagenicity
assessment. The solvent systems were freshly prepared for
each run and the TLC plates were allowed to develop in the
dark for about 0.75-1 hour, usually resulting in 13-14 cm
movement of the solvent front. The plates were then air-dried
and examined for fluorescence under a Chromato-Vue cabinet
model CC-20G (Ultra-Violet Products, Inc., San Gabriel, CA)
using long wavelength UV detection. A band from the aqueous
chlorine and tryptophan reaction products was re-developed on
another TLC plate by using a mobile phase containing hexane:
methylene chloride:acetic acid (50:50:2, v/v/v).
Any bands obtained were scraped off with a spatula,
extracted with acetone and filtered through a Whatman No. 4
qualitative filter paper. As before, all extractions were
performed under yellow lights to ensure that any
photosensitive materials were not destroyed. The filtrate was
finally concentrated by rotary evaporation, weighed and
dissolved in DMSO and used for genotoxicity evaluation. The
stability of the TLC extracts was also checked by leaving some
plates at room temperature for up to 48 hours and monitoring
their long wavelength UV absorbance.

91
Product distribution in the TLC fractions. Aliquots from the
radioactive reaction products (7:1 ratio) were spotted on TLC
plates and developed with the solvents for aqueous chlorine
and chlorine dioxide as described above. The fluorescent
bands were scraped off and dissolved in Scintiverse II
cocktail solution. To ensure that all the radioactivity was
accountered for, the gaps between the fluorescent samples all
the way to the solvent front were also scraped off
individually and dissolved in Scintiverse II cocktail.
Radioactivity in all the samples including silica gel blanks
was counted as previously described.
Genotoxicitv Assays
The mutagenic potentials of all the extracts and
subfractions were evaluated using the Ames Salmonella/
mammalian microsome assay (Ames et al. , 1975). The highly
mutagenic TLC subfractions were further evaluated for
genotoxic effects using the sister chromatid exchange (SCE)
assay in vitro in Chinese hamster ovary (CHO) cells.
Ames Salmonella/mammalian microsome assay
The standard methods developed by Ames et al. (1975) and
later modified by Marón and Ames (1983) were used to determine
the mutagenic activity of the reaction products. Salmonella
typhimurium strains TA98 and TA100 were generously provided
by Dr. Bruce N. Ames (University of California, Berkeley, CA) .

92
The bacteria were grown in nutrient broth (Nutrient Broth No.
2, Oxoid Ltd., Hants, England) and stored as described by
Marón and Ames (1983). The tests were performed with and
without the rat hepatic S-9 mix. The liver S-9 fraction was
prepared from the livers of male Sprague-Dawley rats induced
with Aroclor 1254 according to the recommendations of Marón
and Ames (1983) . The rat liver S-9 preparation was thawed
immediately before use and mixed with an NADPH-generating
system comprising 4 mM NADP, 5 mM glucose-6-phosphate, 8 mM
MgCl2, 33 mM KC1 and 100 mM sodium phosphate (pH 7.4). This
system provided a broad spectrum of xenobiotic metabolizing
enzymes capable of activating or inactivating the test
compounds.
For the regular plate incorporation assay, 0.1 mL of a
fresh overnight bacterial culture (approximately 1 x 108
cells) was added together with predetermined doses of the test
compounds to 2.5 mL of soft top agar containing 10% of 0.5 mM
L-histidine.HC1 and 0.5 mM biotin (Sigma Chemical Co., St.
Louis, MO). In tests requiring metabolic activation, 0.5 mL
of the S-9 mix was also added, while in tests without
metabolic activation it was replaced with 0.5 mL of 0.25 M
sodium phosphate buffer (pH 7.4). The resulting liquid
suspension was thoroughly mixed and poured onto petri dishes
with Vogel-Bonner agar medium containing sources of carbon and
nitrogen for the growth and expression of revertant colonies.

93
Concurrent positive and solvent controls were included
in all experiments to confirm desired reversion properties of
each strain of bacteria and also S-9 activity. In the
presence of the S-9 mix, 2-aminofluorene (2-AF, Aldrich
Chemical Co., Inc., Milwaukee, WI) was used as the positive
control for both strains of bacteria while 2-nitrofluorene
(2-NF, Aldrich Chemical Co.) and methyl methanesulfonate (MMS,
Aldrich Chemical Co.) were used for TA98 and TA100,
respectively, when the S-9 mix was absent. Solvent controls,
mainly DMSO, S-9 mix and sodium phosphate buffer (pH 7.4),
were tested without the addition of test compounds in order
to determine the number of spontaneous colonies for both
tester strains. Four plates were used for each strain and
dose of test chemical or controls and each test sample was
assayed at least twice. The plates were incubated in the
inverted position in a Gravity convection incubator (Precision
Scientific, Chicago, IL) at 37°C for 48 hours. The number of
revertant colonies was counted and recorded as a function of
volume or weight of test sample assayed. The general protocol
complied with the workshop recommendations on the Ames
Salmonella/microsome assay regarding basic techniques such as
strain maintenance, genotypic characteristics checks, S-9
preparation and the examination of the background lawn for
toxicity (de Serres and Shelby, 1979) . The mutagenicity
ratio, the ratio of the number of revertants per test sample
divided by that of the controls, was used for statistical

94
analysis. A ratio of 2 or more is generally considered
positive in this assay (Ames et al., 1975). However, a
modification of the Student's t-test was used to determine
statistical significance (Steel and Torrie, 1980).
Sister chromatid exchange (SCE) assay
Chinese hamster ovary (CHO-Kj-BH*) cells were kindly
provided by Dr. Abraham W. Hsie, Oak Ridge National
Laboratory, Oak Ridge, TN. The cells were grown as monolayers
in Corning T75-cm2 flasks in a Narco incubator (Portland, OR)
with humidified atmosphere of 5% C02 in air at 37°C, in Eagles
medium (Gibco Laboratories, Grand Island, NY) supplemented
with 10% fetal calf serum, 1% glutamine and 1% penicillin-
streptomycin (Gibco Laboratories). To maintain karyotypic
stability, the cells were used no more than 15 passages after
they were received. Cells were usually retrieved from frozen
samples stored in liquid nitrogen and maintained as before.
The assays were performed without the metabolic
activation enzyme system from rats pretreated with Aroclor
1254. The metabolic activation system was omitted because
results from the previously mentioned Ames mutagenicity assay
indicated that it was not required for mutagenic activity.
For the SCE assays cells were grown in Corning T25-cm2 flasks.
One day after culture inoculation, medium was replaced and the
exponentially-growing cells (about 4 x 105 cells/T25-cm2 flask)
obtained by counting aliquots of the cell suspension on a
hemacytometer, were treated with different concentrations of

95
the test chemicals and controls for 2 hours to ensure chemical
interaction with the cells. Eagles basal medium and MMS were
used as negative and positive controls, respectively. After
the exposure period, the media were removed and the cells were
washed twice with phosphate buffered saline (PBS) to remove
any traces of test chemical present. Fresh medium containing
5-bromodeoxyuridine (BrdU, 3 /xg/mL, Sigma Chemical Co.) was
added and incubation was continued for an additional 22 hours.
Cultures were wrapped in aluminum foil and maintained in the
dark with colchicine (0.1 /xg/mL, Sigma Chemical Co.) being
present for the final 2-3 hours of culture.
Chromosome preparations
Three hours after addition of colchicine, cells were
harvested by mitotic shake-off (Terasima and Tolmach, 1961)
and swollen for 20 minutes with a hypotonic solution (75 mM
KC1). Cells were then fixed twice by washing for 20 minutes
each with a non-aqueous fixative (3:1 methanol:glacial acetic
acid, v/v) , then dropped from a distance of 10-15 cm onto
clean cold slides to rupture the cells, and then air-dried.
A modified fluorescence plus Giemsa technique (Goto et
al., 1978; Perry and Wolff, 1974) was used for differential
staining of sister chromatids for SCE analyses. Slides were
stained for 10 minutes in fluorochrome Hoechst 33258 (5 /xg/mL,
Calbiochem-Behring, La Jolla, CA) in Gurr buffer (pH 6.8),
rinsed and mounted in the same buffer, and exposed at 60°C for
1 hour to a UV light source. Slides were rinsed in distilled

96
water, stained in 4% Wright-Giemsa (Diagnostic Systems, Inc.,
Gibbstown, NJ) in Gurr buffer (pH 6.8) and permanently mounted
in Pro-Texx (Lerner Laboratories, New Haven, CT) mounting
medium. Selection of cells for scoring was based on proper
morphology, good spread and a chromosome number of 21 ± 2
(Galloway et al., 1985). SCE frequencies were subjected to
square root transformation to equalize the variances (Latt et
al., 1981) and analyzed statistically using the Dunnett's test
(Steel and Torrie, 1980). A total of 50 metaphases was scored
(25/replicate dose) for each dose tested.
Gas Chromatograph/Mass Spectrometer/Data System
A Finnigan Instruments (Sunnyvale, CA) automated
combination quadrupole mass spectrometer (Model 4500)/gas
chromatograph/data system (Incos Rev. 5.4) was used to
identify some of the mutagens present in the chlorinated
extracts. Mass spectra of solid samples were obtained by use
of the solid probe direct inlet source. Mass spectra of
compounds in the various extracts fractionated by TLC and some
liquid-liquid extracts were obtained and compared to the
spectra of known compounds in the spectral library. A DB-5
capillary column (30 m x 0.32 mm i.d., J. and W. Scientific,
Rancho, Cordova, CA) was programmed linearly from 50-300°C at
a rate of 12°C/minute with helium as the carrier gas. Helium
carrier gas flow rate was maintained at 25 mL/minute.
Chromatograms were run in both positive chemical ionization

97
(Cl) and electron impact (El) modes with methane as the
reagent gas. Electron energies of 70 eV were used to generate
total ion current chromatograms and recorded mass spectra.

CHAPTER IV
RESULTS
Mutagenicity of the Reaction Products of Aqueous
Chlorination of L-Trvptophan
Aqueous chlorine reacted readily with different molar
concentrations of tryptophan at ambient temperature to produce
a dark-colored precipitate. The amount of precipitate formed
increased linearly with time and levelled off after about 5
days. When the molar concentration of aqueous chlorine was
increased more than 15-fold over that of the amino acid, color
formation was not observed.
Rotary evaporation
Preliminary studies were conducted to confirm the
mutagenic activity of chlorination reaction mixtures of
aqueous chlorine and tryptophan and to find the best method
to concentrate and fractionate the reaction products for
genotoxicity assessment. In one study, a reaction mixture
containing equimolar concentrations of aqueous chlorine and
amino acid (0.05 M final concentration) was concentrated about
10-fold by rotary evaporation under vacuum at 55°C. The
concentrate was filtered to obtain a precipitate and a brown
to light-brown filtrate. The filtrate was filter-sterilized
using a 0.45 /¿m membrane filter and the precipitate was
98

99
dissolved in DMSO and assayed for mutagenic activity.
Procedural blanks containing only tryptophan were also
concentrated to serve as controls. The results of this study
are presented in Tables 2 and 3. For statistical analyses of
all the mutagenicity data, the mutagenicity ratio, the ratio
of the number of revertants per test dose (spontaneous and
induced) divided by that of the respective negative controls
(spontaneous revertants), was used. A mutagenicity ratio
value of 2 or more is generally considered as a statistically
significant indication of mutagenic activity (Ames et al.,
1975). Also, a modification of the Student's t-test was used
to determine statistical significance (Steel and Torrie,
1980).
The aqueous concentrate exhibited mutagenic activity
towards both strains of S. typhimurium TA98 and TA100 at all
the doses tested (Tables 2 and 3). There was a decrease in
the number of revertants at the highest doses tested. This
is due to the fact that at these high levels, the compounds
were beginning to show some toxicity to the bacteria.
Addition of the rat hepatic S-9 mix to the test system led to
a decline in mutagenic activity in strain TA100 at all the
doses tested (Table 3). In bacterial strain TA98, there was
a reduction in the mutagenic activity at the lower doses and
an increase in activity at the highest dose tested when the
S-9 mix was added (Table 2). In the latter, addition of the

100
Table 2. Mutagenicity of the aqueous concentrate arising
from the reaction of equimolar concentrations of
aqueous chlorine and tryptophan using Salmonella
tvphimurium strain TA983
Number of revertants/plateb
TA9 8
Sample
Dose/platec
-S9
MRU
+S9
MR
Bacteria
26± 3
281
7
Bacteria + DMSO
25 nL
24± 2
301
7
Aqueous fraction
100 /¿L
225±44
8.7
331
6
1.2*
200 /iL
338±30
13.0
621
30
2.2*
300 ML
256±27
9.8
3811115
13.6
Precipitate
1 mg
41± 5
1.7
361
6
1.2*
5 mg
45± 8
1.9
481
12
1.6
Aqueous fraction6
100 ml
234176
9.0
291
5
1.0*
200 mL
269166
10.3
751
18
2.7
300 /iL
142138
5.5
1621
42
5.8
Tryptophanf
1 mg
271 5
1.1*
331
4
1.1*
5 mg
321 4
1.3
351
4
1.1*
aEquimolar concentrations of aqueous chlorine and tryptophan
(0.0 5M final concentration) were allowed to react in 0.1 M
sodium phosphate buffer at ambient temperature for 24 hours.
The mixture was filtered and the filtrate was concentrated
10-fold by rotary evaporation, filter-sterilized and tested
for mutagenic activity in Salmonella tvphimurium TA98.
Mean ± standard deviations from duplicate runs using four
plates/dose.
cAqueous concentrates were tested as /¿L of sample while the
dry precipitates were tested as mg sample.
Mutagenicity ratio is the number of revertants per test dose
divided by that of the controls. Asterisk denotes not
significantly different from control samples (P=0.05).
eThe concentrate was tested after 7 days storage at 4°C.
fTryptophan solution (0.05 M) was concentrated by rotary
evaporation and and the dry sample was filtered to serve as
blank.

101
Table 3. Mutagenicity of the aqueous concentrate arising
from the reaction of equimolar concentrations of
aqueous chlorine with tryptophan using Salmonella
typhimurium strain TA1006
Sample
Dose/platec
Number
of revertants/plate6
TA100
-S9
MRU
+S9
MR
Bacteria
84± 7
121± 14
Bacteria + DMSO
25 /xL
91± 15
100± 8
Aqueous fraction
100 /xL
850±241
10.1
167± 40
1.4*
200 nL
829± 13
9.9
2221160
1.8*
300 /xL
326± 64
3.9
1051 20
0.9*
Precipitate
1 mg
152± 12
1.7
1551 10
1.5
5 mg
155± 45
1.7
1591 31
1.6
Aqueous fraction6
100 /xl
163± 60
1.9
1351 12
1.1*
200 /xL
3931114
4.7
2731 16
2.3
300 /xL
216± 69
2.6
1211 7
1.0*
Tryptophanf
1 mg
155± 16
1.7
1251 12
1.3
5 mg
168± 21
1.8
1401 9
1.4
aEquimolar concentrations of aqueous chlorine and tryptophan
(0.05 M final concentration) were allowed to react in 0.1 M
sodium phosphate buffer at ambient temperature for 24 hours.
The mixture was filtered and the filtrate was concentrated
10-fold by rotary evaporation, filter-sterilized and tested
for mutagenic activity in Salmonella typhimurium TA100.
^ean ± standard deviations from duplicate runs using four
plates/dose.
cAqueous concentrates were tested as /¿L of sample while the
dry precipitates were tested as mg sample.
Mutagenicity ratio is the number of revertants per test
dose divided by that of the controls. Asterisk denotes not
significantly different from control samples (P=0.05).
eThe concentrate was tested after 7 days storage at 4°C.
fTryptophan solution (0.05 M) was concentrated by rotary
evaporation and the dry sample was filtered to serve as
blank.

102
S-9 mix caused a reduction in the toxicity and thus allowed
the actual number of revertants to be expressed.
The stability of the aqueous concentrate was checked by
storing the samples in a refrigerator at 4°C for 7 days and
then assaying for mutagenic activity. Some loss of mutagenic
activity was observed in strain TA98 after the storage period
(Table 2). In strain TA100, almost all the samples exhibited
major losses in mutagenic activity. This can be seen from the
mutagenicity ratio values in Table 3. The mutagenic compounds
detected in these studies are mainly direct-acting mutagens
which can cause both frameshift (TA98) and base-pair
substitution (TA100) mutations.
Unlike the filtrate, the dark-colored precipitate and
the tryptophan controls failed to exhibit mutagenic activity
in both bacteria strains at all the doses tested (up to 5 mg
dry sample/plate) . Addition of the S-9 mix to the assay
mixture did not have any observable effects on the
mutagenicity of these samples (Table 2 and 3) . All the
positive controls, MMS, 2-AF and 2-NF were mutagenic to both
tester strains at the doses tested in all the mutagenicity
studies. The net number of revertants for these controls
ranged from 4000-6000 revertants/plate.
Liquid-liquid extraction
In another study, the pH of the reaction mixture was
adjusted with HC1 and NaOH to give acidic, neutral and
alkaline fractions. Each fraction was extracted separately

103
with hexane, methylene chloride and ethyl ether. The organic
solvents were removed by rotary evaporation and the residues
evaluated separately for mutagenic activity in the Ames assay.
The results of these studies are presented in Tables 4 and 5.
All three organic solvents extracted some colored substances
from the reaction mixture, which were readily soluble in DMSO.
Upon drying, the methylene chloride and ethyl ether extracts
appeared dark blue to black while the hexane extracts appeared
reddish in color. In some cases the amount of sample
recovered was so little that it was not enough to cover the
full range of doses designed to be analyzed.
The methylene chloride and ethyl ether extracts of the
acidic fraction were found to be mutagenic to S. tvphimurium
strain TA98 in the absence of the S-9 mix while the hexane
extract was not (Table 4). When the S-9 mix was added, there
was a significant decrease in mutagenic activity such that
mutagenicity was negligible. On a comparative basis, the
methylene chloride extracts were more potent than the ethyl
ether extracts at this pH for TA98. At a dose of 500
jug/plate, the methylene chloride extracts showed a relative
mutagenicity value of 5.4, while it was 3.7 for the ethyl
ether at 5 mg/plate (Table 4).
Toward bacteria strain TA100, only the ethyl ether
extract was mutagenic at a rather high dose of 5 mg/plate
(Table 5) . As before, mutagenic activity was decreased in
the presence of the S-9 mix. The acidic aqueous solution

104
Table 4. Mutagenicity of the liquid-liquid extracts of the
reaction products of aqueous chlorination of
tryptophan using Salmonella typhimurium strain
TA98a
Number of revertants/plateb
TA98
Sample
Dose/plate
-S9
MR1"
+S9
MR
Bacteria
31± 6
351
8
Bacteria + DMSO
25 jliL
31± 4
331
7
Acidic fraction
Hexane extract
500 nq
25± 2
0.8*
251
6
0.8*
2 mg
55±17
1.8
381
6
1.2*
Methylene chloride
500 /¿g
167±13
5.4
571
2
1.7
extract
Ether extract
500 /xg
55± 8
1.8
391
3
1.2*
5 mg
114114
3.7
601
7
1.8
Aqueous fraction0
100 /¿L
Te
T
300 /¿L
T
T
Neutral fraction
Hexane extract
250 fig
721 6
2.3
78112
2.4
500 Mg
T
T
1 mg
T
T
5 mg
T
T
Methylene chloride
250 /ig
881 9
2.8
581
7
1.8
extract
500 Mg
871 8
2.8
821
3
2.5
1 mg
1351 7
4.4
170119
5.2
5 mg
T
T
Ether extract
250 Mg
247111
8.0
1031
8
3.1
500 Mg
T
T
1 mg
T
T

105
Table 4-continued.
Number
of revertants/plateb
TA98
Sample
Dose/plate
-S9
MR
+S9
MR
Alkaline fraction
Hexane extract
500 /¿g
35± 8
1.1*
4 3±11
1.3*
Methylene chloride
extract
500 /jg
17± 3
0.5*
90±14
2.7
Ether extract
500 /ng
5 mg
62± 6
88±11
2.0
2.8
37± 5
98±11
1.1*
3.0
Aqueous fraction
100 iiL
300 mL
47± 4
4 6± 6
1.5
1.5
38± 1
NDf
1.2*
aEquimolar concentrations of aqueous chlorine and tryptophan
(final concentration 0.05 M) were allowed to react for 24
hours at room temperature. The reaction mixture was extracted
three times with equal volumes of hexane, methylene chloride
and ethyl ether to obtain neutral organic extracts. The
aqueous solution was split in half and the pH was adjusted
to obtain acidic (pH 1.2) and alkaline (pH 12) fractions.
These two fractions were then subjected to similar solvent
extractions as previously described. The solvent extracts
were concentrated by rotary evaporation and the residues were
weighed and taken up in DMSO for the Ames test using
Salmonella tvphimurium strain TA98.
'’Means of duplicate runs of four plates/dose plus standard
deviation.
cMutagenicity ratio is the number of revertants per test dose
divided by that of the controls. Asterisk denotes not
significantly different from control samples (P=0.05).
dThe acidic and alkaline aqueous fractions remaining after
the solvent extractions were concentrated by rotary
evaporation and tested for mutagenic activity.
eToxic
fNot determined.

106
Table 5. Mutagenicity of the liquid-liquid extracts of the
reaction products of aqueous chlorination of
tryptophan using Salmonella tvphimurium strain
TA100a
Number of revertants/plateb
TA100
Sample
Dose/plate
-S9
MR1"
+S9
MR
Bacteria
13 6± 17
13 0± 16
Bacteria + DMSO
25 /iL
13 5± 14
12 6± 17
Acidic fraction
Hexane extract
500 /ug
137± 18
1.0*
161± 18
1.3
2 mg
198± 17
1.5
170± 6
1.3
Methylene chloride
extract
500 jug
223± 23
1.7
247± 21
2.0
Ether extract
500 jug
167± 31
1.2*
160± 12
1.3
5 mg
306± 37
2.3
211± 5
1.7
Aqueous fraction0
100 /uL
Te
T
300 /uL
T
T
Neutral fraction
Hexane extract
250 /ug
182± 11
1.3
174± 14
1.4
500 jug
T
4231108
3.4
1 mg
T
5031 46
4.0
5 mg
T
T
Methylene chloride
250 jug
141± 16
1.0*
991 11
0.8*
extract
500 /ug
269± 27
2.0
1901 15
1.5
1 mg
493± 57
3.7
3771 29
3.0
5 mg
T
6661 66
5.3
Ether extract
250 /ug
10941152
8.1
1941 11
1.5
500 /ug
T
13261 77
11.0
1 mg
T
T

107
Table 5-continued.
Number of revertants/plateb
TA100
Sample Dose/plate -S9 MR +S9 MR
Alkaline fraction
Hexane extract
Methylene chloride
extract
Ether extract
Aqueous fraction0
500
Mg
77±
6
500
Mg
118±
9
500
Mg
147±
18
5
mg
373±
49
100
IJ. L
163±
19
300
/liL
149±
8
0.6*
180±
11
1.4
0.9*
157±
25
1.2*
1.1*
150±
10
1.2
2.8
2 31±
17
1.8
1.2*
13 6±
12
1.1*
1.1*
NE
ff
aEquimolar concentrations of aqueous chlorine and tryptophan
(final concentration 0.05 M) were allowed to react for 24
hours at room temperature. The reaction mixture was extracted
three times with equal volumes of hexane, methylene chloride
and ethyl ether to obtain neutral orqanic extracts. The
aqueous solution was split in half and the pH was adjusted
to obtain acidic (pH 1.2) and alkaline (pH 12) fractions.
These two fractions were then subjected to similar solvent
extractions as previously described. The solvent extracts
were concentrated by rotary evaporation and the residues
weiqhed and taken up in DMSO for the Ames test using
Salmonella typhimurium strain TA100.
hMeans of duplicate runs of four plates/dose plus standard
deviation.
cMutagenicity ratio is the number of revertants per test dose
divided by that of the controls. Asterisk denotes not
significantly different from control samples (P=0.05).
dThe acidic and alkaline aqueous fractions remaining after
solvent extractions were concentrated by rotary evaporation
and tested for mutagenic activity.
eToxic
fNot determined.

108
remaining after the liquid-liquid extractions was also
concentrated by rotary evaporation and tested for mutagenic
activity but was found to be too toxic to both tester strains
of bacteria. Addition of the S-9 mix to the test system did
not decrease the toxicity (Tables 4 and 5).
At neutral pH, mutagenic activity was detected in all
three solvent extracts toward both strains in the presence or
absence of the S-9 mix. However, the overall mutagenic
activity was lower in the presence of the S-9 mix (Tables 4
and 5) . Toward TA9 8, the ethyl ether and hexane extracts were
mutagenic at 2 50 /z extract was mutagenic over a wide range of doses (Table 4) .
Higher doses of all the extracts in this fraction were highly
toxic to strain TA98, and toxicity was not diminished even in
the presence of the S-9 mix (Table 4) . On the contrary,
toxicity was reduced in strain TA100 in some but not all the
levels when the S-9 mix was added (Table 5).
With regards to the alkaline or basic fraction, only the
ethyl ether extract was positive in tester strain TA98 with
or without the metabolic activation system (Table 4). Toward
TA100, this fraction was mutagenic only in the absence of the
S-9 mix (Table 5) . The degree of mutagenicity in this extract
increased slightly in TA98 but decreased in TA100 when the S-9
mix was present (Tables 4 and 5) . The hexane and methylene
chloride extracts were non-mutagenic at 500 ¿xg/plate except
for a single dose of the latter extract which was mutagenic

109
toward bacteria strain TA98 only in the presence of the S-9
mix (Table 4) . The aqueous solution remaining after the basic
compounds had been extracted was not mutagenic to either
strain with or without the S-9 mix. From these studies, it
was perceived that the most potent mutagens were present as
neutral compounds in the reaction mixture even though some
mutagenicity was present in the acidic and alkaline fractions
as well. Solvent controls of methylene chloride, hexane and
ether were treated the same way and the residues were
dissolved in DMSO and assayed for mutagenic activity. None
of the solvents was found to be mutagenic in the Ames assay.
Based on the above results, the next series of
experiments using only aqueous chlorine was performed at
neutral pH using a 3:1 molar ratio of chlorinating agent to
tryptophan. This ratio was chosen because during food
processing operations amino acid content of process water is
likely to be much higher than in natural waters, and excess
chlorine is used to treat and disinfect process water for
re-use or discharge into the environment (Robinson et al.,
1981) . Reactions were conducted as previously described and
extracted with the three organic solvents as before. The
results of this study are presented in Tables 6 and 7. As
before, mutagenic activity was detected in both tester strains
in the methylene chloride and ethyl ether extracts (Tables 6
and 7). Toward TA98, the ethyl ether extract was very potent
and exhibited a dose related increase in mutagenic activity

110
Table 6. Mutagenicity of the liquid-liquid extracts of the
reaction products arising from the chlorination of
tryptophan using Salmonella typhimurium strain
TA98a
Sample
Dose/plate
Number of revertants/plateb
TA98
-S9
MR1"
+S9
MR
Bacteria
17± 3
22± 4
Bacteria + DMSO
25 fiL
18± 4
22± 5
Hexane extract
500 fig
21± 8
1.2*
32± 6
1.5
1 mg
NDd
ND
5 mg
Te
T
Methylene chloride
500 fig
26± 9
1.4*
41± 8
1.9
extract
1 mg
31± 3
1.7
42± 9
1.9
5 mg
58±10
3.2
42±10
1.9
Ether extract
500 fig
13 0± 9
7.2
53± 2
2.4
1 mg
276±22
15.3
98± 7
4.5
2.5 mg
293±37
16.3
111± 5
5.0
5 mg
T
T
aA 3:1 molar ratio of aqueous chlorine (0.1 M) and tryptophan
(0.03 M) in phosphate buffer (pH 7) were allowed to react at
room temperature for 24 hours. The reaction mixture was
extracted three times each with equal volumes of hexane,
methylene chloride and ethyl ether. The organic solvents were
removed by rotary evaporation and the residues were weighed
and dissolved in DMSO and used for the Ames assay in
Salmonella typhimurium strain TA98.
Results of duplicate runs of 4 plates/dose. Means and
standard deviations.
cMutagenicity ratio is the number of revertants per test dose
divided by that of the controls. Asterisk denoted not
significantly different from control samples (P=0.05).
dNot determined.
eToxic.

Ill
Table 7. Mutagenicity of the liquid-liquid extracts of the
reaction products arising from the chlorination of
tryptophan using Salmonella tvphimurium strain
TA100a
Sample
Dose/plate
Number of revertants/plateb
-S9
TA100
MRe +S9
MR
Bacteria
133± 9
128112
Bacteria + DMSO
25 nL
13 3± 5
104112
Hexane extract
500 nq
138± 15
1.0*
84116
0.8*
1 mg
NDd
ND
5 mg
Te
T
Methylene chloride
500 nq
125± 9
0.9*
170135
1.6
extract
1 mg
246± 14
1.8
1951 9
1.9
5 mg
550± 82
4.1
281122
2.7
Ether extract
500 M9
1108± 54
8.3
303136
2.9
1 mg
15701128
11.8
485154
4.7
2.5 mg
18331112
13.8
899155
8.6
5 mg
T
T
aA 3:1 molar ratio of aqueous chlorine (0.1 M) and tryptophan
(0.03 M) in phosphate buffer (pH 7) were allowed to react at
room temperature for 24 hours. The reaction mixture was
extracted three times each with equal volumes of hexane,
methylene chloride and ethyl ether. The organic solvents were
removed by rotary evaporation and the residues were weighed
and dissolved in DMSO and used for the Ames assay in
Salmonella tvphimurium strain TA100.
Results of duplicate runs of 4 plates/dose. Means and
standard deviations.
cMutagenicity ratio is the number of revertants per test dose
divided by that of the controls. Asterisk denoted not
significantly different from control samples (P=0.05).
“Not determined.
eToxic.

112
until at some higher dose where toxicity was encountered
(Table 6) . Again, the addition of the S-9 mix to the test
system led to a more than 2-fold decrease in mutagenic potency
of this extract (Table 6) . The methylene chloride extract was
only weakly mutagenic in TA98 with the activity being
abolished in the presence of the S-9 mix (Table 6). Both the
ethyl ether and methylene chloride extracts showed dose
related increases in mutagenic activity in TA100 with the
potencies decreasing in the presence of the S-9 mix (Table
7) . The hexane extracts were either nonmutagenic overall, or
were toxic to the bacteria at the doses tested (Tables 6 and
7) .
On a comparative basis, the potency of the 3:1 extracts
were much lower than the 1:1 extracts on a dose to dose basis
(Tables 4 and 6). For example at the 3:1 ratio, a linear
dose-response mutagenic activity curve was obtained for the
ethyl ether extracts at doses of 500 ¡ig up to 2.5 mg/plate
(Tables 6 and 7) while the same does not hold true for the 1:1
extracts. For the 1:1 ratio, a minimum dose of 250 /xg/plate
gave results comparable to a dose of 2.5 mg/plate in the 3:1
ratio. The overall reduction in the mutagenic activity was
less and was probably caused by the formation of less toxic
compounds at the 3:1 ratio, or due to the formation of
products which are not extractable with the organic solvents
used. Results of the liquid-liquid extractions also indicate
that ethyl ether is the most suitable organic solvent for the

113
extraction of the relatively non-polar reaction mixtures of
aqueous chlorination of tryptophan.
Mutagenicity of the Reaction Products of Aqueous
Chlorine or Chlorine Dioxide with L-Trvptophan
The next series of experiments was conducted to compare
the mutagenicities of the reaction products of aqueous
chlorine or chlorine dioxide with tryptophan. Lower
tryptophan concentrations (7 and 5 mM final concentrations)
were selected such that they would be representative of those
amounts likely to be encountered in food processing
operations. The ratios of aqueous chlorine or chlorine
dioxide to amino acid, 7:1, 3:1 and 1:1, were also chosen to
cover the range of concentrations likely to be encountered in
the real world situation. Samples were prepared as depicted
in Fig. 6 and the concentrates evaluated for mutagenic
activity.
Liquid-liquid extraction
Aqueous chlorine. The results of the liquid-liquid extraction
of the aqueous chlorine-tryptophan reaction products using
ethyl ether as the extracting solvent are presented in Tables
8 and 9. The reaction products of aqueous chlorine with
tryptophan were mutagenic to TA98 without metabolic activation
at the 1:1 ratio. Mutagenic activity was abolished at the 3:1
ratio and increased dramatically at the 7:1 ratio (Table 8).
Higher doses of the 3:1 and 7:1 ratios were toxic to the

114
Table 8. Mutagenicity of the ethyl ether extracts arising
from the aqueous chlorination of tryptophan using
Salmonella tvphimurium strain TA98a
Number
of revertants/plateb
TA98
Sample
Dose/plate
—S9
MRC
+S9
MR
Bacteria
2 0± 6
29± 3
Bacteria + DMSO
25 /iL
21± 3
28± 4
1:1 Extract
1 mg
42± 9
2.0
3 6± 5
1.3
2 mg
58± 6
2.8
47±10
1.7
5 mg
68±12
3.2
42±11
1.5
3:1 Extract
250 nq
29± 5
1.4
3 6± 5
1.3
1 mg
38± 2
1.8
47± 5
1.7
5 mg
Td
T
7:1 Extract
500 nq
174±18
8.3
68± 7
2.4
1 mg
237±16
11.3
92± 2
3.3
5 mg
T
285± 8
10.2
aThree molar ratios of aqueous chlorine to tryptophan (1:1,
3:1 and 7:1) were allowed to react at room temperature for 24
hours. The reaction products from each mixture were extracted
separately with ethyl ether and concentrated for mutagenicity
assessment in Salmonella tvphimurium strain TA98.
Results of duplicate runs of 4 plates/dose. Means plus
standard deviations.
cMutagenicity ratio is the number of revertants per test dose
divided by that of the controls. Asterisk denotes not
significantly different from control samples (P=0.05).
dToxic.

115
Table 9. Mutagenicity of the ethyl ether extracts arising
from the aqueous chlorination of tryptophan using
Salmonella tvphimurium strain TAIOO3
Number
of revertants/plateb
TA100
Sample
Dose/plate
-S9
MR1"
+S9
MR
Bacteria
123± 9
12 0±
9
Bacteria + DMSO
25 /¿L
124± 9
124±
5
1:1 Extract
1 mg
106± 9
0.9*
126±
27
1.0*
2 mg
156±2 0
1.3
151±
9
1.2
5 mg
134±10
1.1*
217±
23
1.8
3:1 Extract
1 mg
179±37
1.4
14 3±
7
1.2
2 mg
106±21
0.9*
14 6±
13
1.2
5 mg
Td
T
7:1 Extract
1 mg
667±51
5.4
407±
32
3.3
2 mg
706±71
5.7
644±
59
5.2
5 mg
T
1203±123
9.7
aThree molar ratios of aqueous chlorine to tryptophan (1:1,
3:1 and 7:1) were allowed to react at room temperature for 2 4
hours. The reaction products from each mixture were extracted
separately with ethyl ether and concentrated for mutagenicity
assessment in Salmonella tvphimurium strain TA100.
Results of duplicate runs of 4 plates/dose. Means plus
standard deviations.
cMutagenicity ratio is the number of revertants per test dose
divided by that of the controls. Asterisk denotes not
significantly different from control samples (P=0.05).
dToxic.

116
bacteria, while those of the 1:1 ratio were not. Addition of
the S-9 mix to the test system led to a decline in the
mutagenic activity of all the treatments and decreased the
toxicity in the 7:1 ratio. However, the addition of S-9 mix
did not decrease the toxicity observed in the 3:1 ratio (Table
8) .
Toward bacteria strain TA100, only the 7:1 ratio was
mutagenic with mutagenic activity decreasing in the presence
of the S-9 mix (Table 9). As before, higher concentrations
of the 7:1 and 3:1 ratios were toxic to the bacteria.
Toxicity in the 7:1 and not the 3:1 ratio decreased in the
presence of the S-9 mix and the actual number of revertants
was expressed. The results in Tables 8 and 9 indicate that
the ethyl ether extractable reaction products are mainly
frameshift mutagens with greater potency towards TA98 and are
also capable of causing base-pair substitution mutations in
TA100.
Chlorine dioxide. A similar pattern was observed for the
reaction products of chlorine dioxide with tryptophan. The
reaction products for the three ratios showed mutagenic
activity in tester bacteria TA98 in the absence of the S-9
mix (Table 10) . However, the overall mutagenic activity
decreased slightly from the 1:1 to the 3:1 ratio and increased
again in the 7:1 ratio for this bacteria strain. Addition of
the S-9 mix led to a decline in all the ratios except at the
1 mg/plate dose of the 3:1 ratio (Table 10). In the case of

117
Table 10. Mutagenicity of the ethyl ether extracts arising
from the reaction of aqueous chlorine dioxide
with tryptophan using Salmonella typhimurium strain
TA98a
Number of revertants/plateb
TA98
Sample
Dose/plate
-S9 MR0 +S9 MR
Bacteria
2 5± 3
23± 4
Bacteria + DMSO
25 fJLh
21± 3
22± 7
1:1 Extract
1 mg
63±10
3.0
30± 3
1.4*
2 mg
lilt 6
5.3
64± 9
3.0
5 mg
168±14
8.0
152±19
7.2
3:1 Extract
1 mg
34±10
1.6
112± 4
5.3
2 mg
126±37
6.0
95±11
4.5
5 mg
112± 8
5.3
101±10
4.8
7:1 Extract
1 mg
107±16
5.1
57± 8
2.7
2 mg
163±2 5
7.8
98±14
4.7
5 mg
250±3 3
11.9
141± 5
6.7
aThree molar ratios of aqueous chlorine dioxide to tryptophan
(1:1, 3:1 and 7:1) were allowed to react at room temperature
for 24 hours. The reaction products from each mixture were
extracted separately with ethyl ether and concentrated for
mutagenicity assessment in Salmonella typhimurium strain TA98.
Results of duplicate runs of 4 plates/dose. Means plus
standard deviations.
cMutagenicity ratio is the number of revertants per test dose
divided by that of the controls. Asterisk denotes not
significantly different from control samples (P=0.05).

118
S. tvphimurium strain TA100, increasing the molar ratio of
chlorine dioxide from 1 to 7 led to an overall increase in the
mutagenic activity, especially at the highest doses tested
(Table 11). Again the addition of the rat hepatic S-9 mix led
to a decline in the potency of some but not all the extracts.
Also, mutagenic activity was not abolished at all the doses
tested (Table 11). Looking at the ratio where both produced
mutagens, it is clear that aqueous chlorine reaction products
were more potent than those of aqueous chlorine dioxide
(Tables 8 and 10) . These results indicate that both chlorine
dioxide and aqueous chlorine are capable of producing potent
frameshift mutagens when reacted with tryptophan. No toxicity
was observed in the ethyl ether extracts of chlorine dioxide-
tryptophan reaction mixtures even at very high doses of up to
5 mg/plate (Tables 10 and 11). However, similar doses of the
aqueous chlorine reaction products were mostly toxic
especially at the higher chlorine to tryptophan ratios (Tables
8 and 9). Also ethyl ether is a suitable organic solvent
capable of extracting some but not all of the mutagens present
in the aqueous reaction mixtures.
Amberlite XAD adsorption
Distribution of radioactivity. Preliminary experiments were
conducted to find the best method for concentrating the
chlorination reaction mixtures using Amberlite XAD-2 and XAD-8
either separately in different columns or in combination in
one column. The results of this study indicated that the

119
Table 11. Mutagenicity of the ethyl ether extracts arising
from the reaction of aqueous chlorine dioxide
with tryptophan using Salmonella typhimurium strain
TA100a
Number
of revertants/plateb
TA100
Sample
Dose/plate
—S9
MRg
+S9
MR
Bacteria
135±11
1231 3
Bacteria + DMSO
25 nL
14 0±14
1181 8
1:1 Extract
1 mg
182±13
1.3
159120
1.3
2 mg
371±42
2.7
216112
1.8
5 mg
599117
4.3
497190
4.2
3:1 Extract
1 mg
176121
1.3
163118
1.4
2 mg
342119
2.4
257123
2.1
5 mg
659156
4.7
567155
4.8
7:1 Extract
1 mg
252113
1.8
244132
2.1
2 mg
449164
3.2
3301 4
2.8
5 mg
844167
6.0
605157
6.7
aThree molar ratios of aqueous chlorine dioxide to tryptophan
(1:1, 3:1 and 7:1) were allowed to react at room temperature
for 24 hours. The reaction products from each mixture were
extracted separately with ethyl ether and concentrated for
mutagenicity assessment in Salmonella typhimurium strain
TA100.
Results of duplicate runs of 4 plates/dose. Means plus
standard deviations.
cMutagenicity ratio is the number of revertants per test dose
divided by that of the controls.

120
recovery of the reaction products was irrespective of whether
XAD-2 resins were placed on top of the XAD-8 resins or vice
versa (Table 12). In both cases, using the two resins in one
column provided better recoveries than using XAD-2 alone
(Table 12). When XAD-8 was placed on top of XAD-2, the
recoveries were slightly higher (48% against 46%), but again,
the differences were not statistically significant (Table 12) .
Data on the acidification of the reaction mixture prior to
Amberlite XAD adsorption are presented in Table 13. Maximum
recovery of the reaction products was obtained at pH 2.5 (60%
recovery) than at pH 6.0 (45% recovery) (Table 13). Also, at
any given pH, product recovery was independent of whether
aqueous chlorine or chlorine dioxide was used (Table 13).
The amount of radioactivity recovered was less for
reaction mixtures with excess chlorine or chlorine dioxide
(7:1 molar ratio) than those at the 1:1 ratio. However, the
eluates in the aqueous chlorine and chlorine dioxide reaction
mixtures at the 7:1 ratio were not significantly different
from each other (Table 14).
Mutagenicity of the Amberlite XAD eluates
Control solutions. Procedural blanks containing aqueous
chlorine, chlorine dioxide or tryptophan alone in 0.1 M sodium
phosphate buffer were mixed for 24 hours as described for the
actual samples. After reaction, all the samples including the
tryptophan solution were dechlorinated with sodium sulfite.
The control blanks were then concentrated by resin adsorption,

121
Table 12. Percent distribution of radioactivity after resin
adsorption in the reaction of aqueous chlorine with
tryptophan3
Sample
fraction
XAD-8/2b
XAD-2/8b
XAD-2
Filtratec
100
100
100
Column
effluent
48.912.7
43.310.5
54.211.6
Column
eluated
48.0±0.6X
45.511.7X
37.510.5y
Loss6
3.1±0.1
11.210.3
8.310.1
aAqueous chlorine was allowed to react with a radiolabeled
tryptophan solution (1:1 molar ratio) for 4 hours. The
reaction mixture was filtered and concentrated by resin
adsorption.
bXAD-8/2 is equivalent to placinq XAD-8 on top of XAD-2 (1:1
mixture) and vice versa.
cAfter filtration, there was about 20% loss of radioactivity.
Therefore the filtrate was assumed to contain 100% product
prior to resin adsorption.
“^Means with the same letter in the same row are not
significantly different from each other (P<0.05).
eThe amount of radioactivity unaccounted for was assumed to
be irreversibly linked to the resins or lost through
volatilization.

122
Table 13. Recovery of radioactivity after resin adsorption in
the reaction of aqueous chlorine and chlorine
dioxide with tryptophan9
pH 6
. 0
pH 2
.5
Sample
Tryptophan +
Tryptophan +
fraction
H0C1
cio2
HOC1
cio2
Filtrate6
100
100
100
100
Column
effluent
48.9±2.7
44.4±2.3
35.7±2.9
3 6.1±0.5
Column
eluatec
48.0±0.6X
44.0±1.4X
56.5±3.ly
60.6±1.8y
Lossd
3.1±0.1
11.6±0.2
7.8±0.2
3.3±0.2
aAqueous chlorine or chlorine dioxide was allowed to react
with a radiolabeled tryptophan solution for 4 hours at room
temperature at pH 6.0. The reaction was filtered and the
mixture divided into two portions. The pH of one-half was
adjusted to 2.5 with 85% phosphoric acid and both fractions
concentrated separately by resin adsorption with Amberlite
XAD-8/2 (1:1 mixture). The adsorbed substances were eluted
successively with acetone, methanol and ethyl ether. The
eluates were concentrated by rotary evaporation, adjusted to
a fixed volume by the addition of chlorine demand-free water,
and aliquots were checked for radioactivity.
bAfter filtration there was about 20% loss of radioactivity.
Therefore the filtrate was considered to be 100%.
cMeans with same letter in the same row are not significantly
different from each other (P<0.05).
^he amount of radioactivity unaccounted for was assumed to
be irreversibly linked to the resins or lost through
volatilization.

123
Table 14. Percent distribution of radioactivity after resin
adsorption in the reaction of aqueous chlorine or
chlorine dioxide with tryptophan3
Sample
fraction
Tryptophan
+ buffer
Tryptophan
+ H0C1
Tryptophan
+ cio2
Filtrate6
100
100
100
Column effluent
35.6±2.1
48.6±2.0
41.2±0.8
Column eluatec
58.4±1. lx
49.5±0.7y
48.6±1.2y
Lossd
6.0±0.2
3.7±0.1
10.2±0.3
aAqueous chlorine, chlorine dioxide or buffer were allowed to
react with a radiolabelled tryptophan solution (7:1 molar
ratio) at room temperature for 24. Any excess chlorine or
chlorine dioxide remaining after reaction was removed by
addition of sodium sulfite and the solutions were filtered
and concentrated by resin adsorption. The adsorbed substances
were successively eluted with acetone, methanol and ethyl
ether. The eluates were concentrated by rotary evaporation
and aliquots checked for radioactivity.
bAfter filtration, the filtrate was assumed to be 100%
starting material. There was about 20% loss of radioactivity
in the precipitate or as volatiles.
cMeans with the same letter in the same row are not
significantly different from each other (P<0.05).
dThe amount of radioactivity that was not accounted for was
assumed to be trapped in the resins or lost through
volatilization.

124
eluted with acetone, methanol and ethyl ether, respectively,
and assayed for mutagenic activity as previously described.
The results indicated that none of the procedural blanks was
mutagenic in either strain of bacteria at the doses tested
(Table 15).
Aqueous chlorine. Sample preparation for the above reactions
are summarized in Fig. 6. Mutagenic activity was detected in
all the Amberlite XAD acetone eluates in strain TA98 at the
three ratios of aqueous chlorine to the amino acid in the
absence of the S-9 mix (Table 16). In all cases, the acetone
eluates contained the most potent mutagens as compared to the
methanol eluates at the three ratios. A dose dependent
increase in mutagenic activity was observed in all these
eluates until at some higher dose when toxicity was
encountered. Again, the degree of mutagenicity increased with
increasing chlorine concentration. For example at a test dose
of 100 /nL/plate, the relative mutagenicity ratios were 4.3,
6.9 and 15.7, respectively, for the 1:1, 3:1 and 7:1 ratios
of aqueous chlorine:tryptophan extracts (Table 16).
The methanol eluates were mutagenic at the 1:1 and 3:1
ratios but not at the 7:1 ratio toward bacteria strain TA98.
The 3:1 ethyl ether eluate was only weakly mutagenic to this
strain of bacteria. No ethyl ether eluates were present in
the 1:1 and 7:1 samples because examination of the column for
color indicated that all the adsorbed substances had been
removed by the acetone and methanol. Also, subsequent elution

125
Table 15. Mutagenicity of Amberlite XAD eluates arising
from control aqueous chlorine, chlorine dioxide
and tryptophan solutions3
Number of revertants/plateb
TA98 TA100
Sample
Dose/plate
-S9
MR
-S9
MR
Bacteria
35±
2
92±15
Bacteria + DMSO
25
ML
3 6±
4
84± 7
Aoueous chlorine
Acetone eluate
100
¿¿L
40±
2
1.1
94±17
1.0*
300
ML
38±
3
1.1*
93± 6
1.0*
Methanol eluate
250
M9
37±
3
1.0*
90±11
1.1*
1
mg
37±
3
1.0*
103±13
1.2
2
mg
38±
1
1.1*
98±10
1.2*
Ether eluate
500
Mg
3 6±
4
1.0*
84±12
1.0*
Chlorine dioxide
Acetone eluate
100
ml
43±
3
1.2
84± 6
0.9*
300
ml
40±
6
1.1*
90±18
1.0*
Methanol eluate
250
ml
38±
2
1.1*
89± 3
1.1*
1
mg
35±
3
1.0*
89± 7
1.1*
5
mg
38±
4
1.1*
99±11
1.2*
Ether eluate
500
ml
2 6±
1
0.7*
84± 5
1.0*
TrvDtoohan
Acetone eluate
100
ml
37±
3
1.1*
8 3±11
0.9*
300
ml
38±
2
1.1*
91± 5
1.0*
Methanol eluate
250
Mg
35±
2
1.0*
89± 8
1.1*
1
mg
36+
3
1.0*
103±16
1.2*
5
mg
38±
2
1.1*
91± 5
1.1*
Ether eluate
500
Mg
3 6±
2
1.0*
98±13
1.2*

126
Table 15-continued.
aAqueous chlorine, chlorine dioxide or tryptophan alone were
mixed in phosphate buffer for 24 hours. After reaction any
available chlorine present in the aqueous chlorine and
chlorine dioxide solutions were removed by the addition of
sodium sulfite. The solutions from each mixture were then
concentrated by resin adsorption and eluted separately with
acetone, methanol and ethyl ether. The eluates were
concentrated separately by rotary evaporation and aliquots
assayed for mutagenic activity in Salmonella tvphimurium
strains TA98 and TA100.
bResults of duplicate runs of 4 plates/dose. Means plus
standard deviations.
cMutagenicity ratio is the number of revertants per test dose
divided by that of the controls. Asterisk denotes not
significantly different from control samples (P=0.05).

127
Table 16. Mutagenicity of Amberlite XAD eluates arising
from the reaction of aqueous chlorine with
tryptophan using Salmonella typhimurium strain
TA98a
Number of revertants/plateb
TA98
Sample Dose/plate -S9 MRC +S9 MR
Bacteria
Bacteria + DMSO
1:1 Acetone eluate
1:1 Methanol eluate
3:1 Acetone eluate
3:1 Methanol eluate
3:1 Ether eluate
7:1 Acetone eluate
7:1 Acetone eluate
precipitate6
2 0± 6
29± 3
25
/xL
21± 3
28± 4
100
M L
8 6±2 5
4.3
52114
1.8
200
/X L
196± 7
9.8
731 8
2.5
300
/x L
Td
T
1
mg
38± 6
1.8
441 7
1.6
2
mg
44± 2
2.1
36112
1.3
5
mg
85±12
4.0
56111
2.0
50
/xL
107±20
5.4
391 6
1.3
100
/xL
138± 9
6.9
481 4
1.7
200
/xL
T
T
1
mg
35± 2
1.7
451 2
1.6
2
mg
60± 8
2.9
501 8
1.8
500
Mg
33± 7
1.6
391 5
1.4
1
mg
42± 3
2.0
501 8
1.8
50
/xL
199±13
10.0
1141 8
3.9
100
/xL
313±3 0
15.7
126128
4.3
200
/xL
T
T
500
Mg
71± 8
3.4
461 6
1.6
2
mg
137±21
6.5
95110
3.4
5
mg
T
T
500
Mg
29± 4
1.4
501 5
1.8
1
mg
3 0± 6
1.4
551 8
2.0
7:1 Methanol eluate

128
Table 16-continued.
aThree molar ratios of aqueous chlorine to tryptophan (1:1,
3:1 and 7:1) were allowed to react at room temperature for 24
hours. The reaction products from each reaction were
concentrated by XAD adsorption and then eluted separately with
acetone, methanol and ethyl ether. The eluates were
concentrated by rotary evaporation and assayed for mutagenic
activity in Salmonella tvphimurium strain TA98.
bResults of duplicate runs of 4 plates/dose. Means plus
standard deviations.
cMutagenicity ratio is the number of revertants per test dose
divided by that of the controls. Asterisk denotes not
significantly different from control samples (P=0.05).
dToxic.
eAfter concentrating the acetone eluate from this ratio, some
precipitate was obtained which was dissolved in DMSO and
tested for mutagenic activity.

129
with ethyl ether provided negative or negligible yields which
could not be assayed.
Addition of the S-9 mix to the test system led to a
decline in the mutagenic activity in all but one of the
eluates. More than a 2-fold decrease in mutagenic activity
was observed in the highly mutagenic acetone eluates at all
the levels and ratios tested. However, in the case of the
7:1 methanol eluate, weak mutagenic activity was detected only
in the presence of the S-9 mix (Table 16). At the 7:1 ratio,
a precipitate was formed upon removing the acetone by rotary
evaporation. The precipitate tested positive in TA98 and the
potency decreased in the presence of the S-9 mix (Table 16).
Toward S. typhimurium TA100, only the 1:1 and 7:1 acetone
eluates and 7:1 precipitate were mutagenic at the levels
tested when the S-9 mix was absent (Table 17). The 7:1
acetone eluate was highly mutagenic at a test dose of 50
/¿L/plate with a mutagenicity ratio of 17.9 Increasing the
doses above 50 /¿L/plate led to toxicity in the bacteria. The
3:1 acetone eluate was not mutagenic but toxic to TA100 at the
highest dose level tested. This lends further credence to the
observation that the mutagenic compounds are mainly frameshift
mutagens. Addition of the S-9 mix to these compounds led to
a decrease in the potency of the 1:1 and 7:1 ratios but
increased potency of the precipitate and the 3:1 acetone
eluate. The ethyl ether and methanol eluates were non-

130
mutagenic in TA100 with or without the rat hepatic enzyme
metabolizing system (Table 17).
Chlorine dioxide. With chlorine dioxide, the acetone
concentrates of the XAD eluates were all mutagenic to TA98 in
the absence of the S-9 mix. Again mutagenic activity
decreased at the 3:1 chlorine dioxide:tryptophan ratio and
increased further at the 7:1 ratio. The mutagenicity ratios
for a test dose of 200 /¿L/plate were 7.1, 3.9 and 8.5,
respectively for the 1:1, 3:1 and 7:1 ratios of chlorine
dioxide:tryptophan reaction products (Table 18) . In the
absence of the S-9 mix, the ethyl ether and methanol eluates
were weakly mutagenic or non-mutagenic. Unlike the 7:1
aqueous chlorine eluate, no precipitate was formed in the 7:1
chlorine dioxide reaction. In the presence of the S-9 mix,
a decrease in mutagenic activity was observed in all the
acetone eluates of the 3 ratios even though mutagenic activity
was not abolished. Also, the 1:1 ethyl ether and the 3:1
methanol eluates became mutagenic on addition of the S-9 mix,
which suggests that these solvents were extracting some
mutagenic compounds from the column which are not direct-
acting but require metabolic activation (Table 18). The only
eluate which showed any toxicity toward TA98 was the 1:1
acetone extract and toxicity was not diminished in the
presence of the S-9 mix.
Toward strain TA100, the acetone eluates of all 3 ratios
of the chlorine dioxide-tryptophan reaction products increased

131
Table 17. Mutagenicity of Amberlite XAD eluates arising
from the reaction of aqueous chlorine with
tryptophan using Salmonella typhimurium strain
TA100a
Number of revertants/plateb
TA100
Sample Dose/plate -S9 MRC +S9 MR
Bacteria
Bacteria + DMSO
1:1 Acetone eluate
1:1 Methanol eluate
3:1 Acetone eluate
3:1 Methanol eluate
3:1 Ether eluate
7:1 Acetone eluate
7:1 Acetone eluate
precipitate6
12 3±
9
25
/iL
124±
9
100
/i L
225±
14
200
/i L
299±
56
300
/iL
Td
500
Mg
141±
8
1
mg
152±
16
2
mg
129±
19
100
ml
188±
30
200
/iL
214±
30
300
/iL
T
1
mg
134±
3
2
mg
156±
11
500
Mg
13 5±
14
1
mg
117±
8
25
/¿L
15561151
50
/iL
2 2 05±
55
100
/iL
T
2
mg
539±
34
5
mg
491±
62
500
Mg
124±
11
1
mg
158±
7
12 0± 9
124± 5
1.8
120116
1.0*
2.4
254± 4
2.1
T
1.1
163121
1.3
1.2
1631 9
1.3
1.0*
1731 3
1.4
1.5
85112
0.7*
1.7
310129
2.6
T
1.1*
1351 4
1.1
1.3
153115
1.2
1.1*
1511 4
1.2
0.9*
161119
1.3
12.7
758153
6.3
17.9
989177
8.2
T
4.3
459143
3.7
4.0
717160
6.0
1.0*
1511 7
1.2
1.3
1771 8
1.4
7:1 Methanol eluate

132
Table 17-continued.
aThree molar ratios of aqueous chlorine to tryptophan (1:1,
3:1 and 7:1) were allowed to react at room temperature for 24
hours. The reaction products from each reaction were
concentrated by XAD adsorption and eluted separately with
acetone, methanol and ethyl ether. The eluates were
concentrated by rotary evaporation and assayed for mutagenic
activity in Salmonella tvphimurium strain TA100.
bResults of duplicate runs of 4 plates/dose. Means plus
standard deviations.
cMutagenicity ratio is the number of revertants per test dose
divided by that of the controls. Asterisk denotes not
significantly different from control samples (P=0.05).
dToxic.
eAfter concentrating the acetone eluate from this ratio, some
precipitate was obtained which was dissolved in DMSO and
tested for mutagenic activity.

133
Table 18. Mutagenicity of Amberlite XAD eluates arising
from the reaction of aqueous chlorine dioxide
with tryptophan using Salmonella tvohimurium
strain TA98a
Number of revertants/plateb
TA98
Sample Dose/plate -S9 MRC +S9 MR
Bacteria
Bacteria + DMSO
1:1 Acetone eluate
1:1 Methanol eluate
1:1 Ether eluate
3:1 Acetone eluate
3:1 Methanol eluate
7:1 Acetone eluate
25± 3
23± 3
25
M L
21± 3
2 0± 7
100
/¿L
121±22
4.8
7 0± 6
3.0
200
ml
177±3 3
7.1
62±16
2.7
300
M L
Td
T
1
mg
41± 3
2.0
38± 5
1.9
2
mg
35±14
1.7
40± 6
2.0
1
mg
18± 4
0.9*
29± 5
1.4*
2
mg
16± 4
0.8*
4 3±11
2.2
50
ml
74± 3
3.0
43± 4
1.9
100
ml
112±13
4.5
58± 4
2.5
200
ml
97±2 7
3.9
62± 8
2.7
1
mg
26+11
1.2*
4 6± 6
2.3
2
mg
31±15
1.5*
56±11
2.8
5
mg
3 6± 6
1.7
110± 3
5.5
100
ml
178±32
7.1
98± 6
4.3
200
ml
213±15
8.5
93±16
4.0
300
ml
161±26
6.4
118±12
5.1
1
mg
25± 4
1.2*
32± 5
1.6
2
mg
3 0± 8
1.4*
34± 7
1.7
5
mg
47± 7
2.2
28± 5
1.4*
7:1 Methanol eluate

134
Table 18-continued.
aThree molar ratios of aqueous chlorine dioxide to tryptophan
(1:1, 3:1 and 7:1) were allowed to react at room temperature
for 24 hours. The reaction products from each reaction were
concentrated by XAD adsorption and eluted separately with
acetone, methanol and ethyl ether. The eluates were
concentrated by rotary evaporation and assayed for mutagenic
activity in Salmonella tvphimurium strain TA98.
bResults of duplicate runs of 4 plates/dose. Means plus
standard deviations.
cMutagenicity ratio is the number of revertants per test dose
divided by that of the controls. Asterisk denotes not
significantly different from control samples (P=0.05).
dToxic.

135
with increasing chlorine dioxide concentrations in the absence
of the S-9 mix. Toxicity was observed in the 1:1 and 3:1
acetone eluates but not in the 7:1 eluates. When the S-9 mix
was present, there was an overall increase in the mutagenic
activity in most of these samples as shown by their relative
mutagenicity ratios (Table 19). The methanol and ethyl ether
eluates were non-mutagenic either in the presence or absence
of the S-9 mix. Again by comparing the relative mutagenicity
ratios, it appears that the mutagens are mainly frameshift
mutagens even though they are also capable of giving rise to
base-pair substitution mutations.
Fractionation of the Reaction Products of Aqueous
Chlorine or Chlorine Dioxide and Tryptophan
Thin layer chromatographic analyses of the chlorination
reaction products
A schematic representation of sample preparation
procedures for the TLC analyses is presented in Fig. 7.
Different solvent systems and solvent combinations were
evaluated for the separation of the acetone eluates of the
Amberlite XAD-2/8 concentrates. The solvents included
chloroform, acetone, methanol, benzene, propanol, acetic acid
and methylene chloride. A solvent system containing hexane,
dichloromethane, ethyl ether, methanol and acetic acid was
found to be suitable for developing the plates. With the 7:1
aqueous chlorine:tryptophan extracts, five separate
identifiable fluorescent bands were obtained under a long

136
Tryptophan + aqueous chlorine or chlorine dioxide
(chlorine to tryptophann ratio 7:1)
24 h at 25 C
Adjust to pH 2.5
\K
Amberlite XAD-2/8 adsorption
Acetone elution
\K
Concentration by rotary evaporation
\|/
Thin layer chromatography detection using long
wavelength UV absorption
V
Acetone extraction of fluorescent bands
V
Concentration by rotary evaporation
V
Assay for genotoxicity
-Ames Salmonella/microsome assay
-Sister chromatid exchange assay
Figure 7. Schematic presentation of sample preparation for
thin layer chromatography and genotoxicity
assessment

137
Table 19. Mutagenicity of Amberlite XAD eluates arising
from the reaction of aqueous chlorine dioxide
with tryptophan using Salmonella typhimurium
strain TA100a
Number of revertants/plateb
TA100
Sample Dose/plate
-S9
MRC
+S9
MR
Bacteria
135±11
123± 3
Bacteria + DMSO
25
/iL
140±10
118± 8
1:1 Acetone eluate
100
ml
3 32±18
2.7
240±14
2.0
200
/iL
391±29
2.9
400±16
3.3
300
/¿L
Td
T
1:1 Methanol eluate
1
mg
152±12
1.1*
142±17
1.2
2
mg
152±12
1.1*
145± 8
1.2
1:1 Ether eluate
1
mg
170±27
1.2*
109± 5
0.9*
2
mg
139± 6
1.0*
103±12
0.9*
3:1 Acetone eluate
100
/iL
463±53
3.4
496128
4.0
200
ml
529±44
3.9
512171
4.2
300
ml
T
T
3:1 Methanol eluate
2
mg
148±12
1.1*
1221 4
1.0*
5
mg
158±13
1.1*
122111
1.0*
7:1 Acetone eluate
50
/iL
371±67
2.7
131137
1.1*
200
/iL
373±23
2.8
512137
4.2
300
/iL
656±69
4.9
1036122
8.4
7:1 Methanol eluate
2
mg
160±28
1.1*
113137
1.0*
5
mg
167±14
1.2
131111
1.1*

138
Table 19-continued.
aThree molar ratios of aqueous chlorine dioxide to tryptophan
(1:1, 3:1 and 7:1) were allowed to react at room temperature
for 24 hours. The reaction products from each reaction were
concentrated by XAD adsorption and eluted separately with
acetone, methanol and ethyl ether. The eluates were
concentrated by rotary evaporation and assayed for mutagenic
activity in Salmonella tvphimurium strain TA100.
bResults of duplicate runs of 4 plates/dose. Means plus
standard deviations.
cMutagenicity ratio is the number of revertants per test dose
divided by that of the controls. Asterisk denotes not
significantly different from control samples (P=0.05).
dToxic.

139
wavelength UV lamp on the TLC plates developed with a
hexane:ethyl ether:acetic acid (50:50:1, v/v/v) solvent
system. These bands were designated band # 1-5. Band #1 with
an Rf value 0 retained some highly polar colored products with
relatively no mobility on the TLC plates. Band #2 with an Rf
value of 0.08 moved very little from the origin and was
composed of mainly polar compounds. These two TLC bands
represented the most polar compounds in the reaction mixture
and due to their greater affinity for the polar stationary
phase, their movements were limited. Bands #3 and 4 with Rf
values of 0.22 and 0.38, respectively, represented compounds
of intermediate polarity and were much better resolved than
the previous bands. The last band, #5, with an Rf value of
0.54 was the least polar of all the five bands. Bands #1, 3
and 4 fluoresced green under the long wavelength UV lamp at
366 nm while bands #2 and 5 fluoresced blue. The fluorescent
intensities of bands 3 and 4 were similar while band 5
fluoresced much more brightly than band #2.
Due to the high mutagenic activity of band #5, it was
further fractionated on another TLC plate using a solvent
system containing hexane, methylene chloride and acetic acid
(50:50:2, v/v/v). The products from the second dimension TLC
fractions were then viewed under the UV lamp to determine the
presence of fluorescent bands. Three major fluorescent bands
could be visualized from the TLC plates which showed both blue
and green fluorescence. The blue fluorescent compounds were

140
designated band 5c with an Rf value of 0.31 and band 5h with
an Rf value of 0.88, respectively. The green fluorescent band
5e had an Rf value of 0.62.
With chlorine dioxide only 4 fluorescent bands could be
visualized under a UV lamp at 366 nm from the TLC plates
developed with the hexane:ethyl ether: methanol:acetic acid
(63:30:7:1, v/v/v/v) solvent system. The fractions also
showed fluorescence in the short wavelength UV range (254 nm)
but of much lower intensity. The bands were designated A, B,
C and D with corresponding Rf values of 0, 0.13, 0.31 and
0.44. The reaction products in these fluorescent bands
appeared to be more polar than those from aqueous
chlorine-treated samples due to their relatively low mobility
on the TLC plates. Bands A and B fluoresced green while C and
D fluoresced blue with D being the most intense.
For both aqueous chlorine and chlorine dioxide reaction
products, there was some loss of fluorescence when the plates
were left in the open at ambient temperature for more than 2
hours and about 90% of the fluorescence disappeared after
about 48 hours. However, fluorescence was maintained for an
extended period of time when the bands were extracted with and
stored in acetone. Judging from the Rf values of the
fluorescent compounds, none of the byproducts of the reactions
between tryptophan and aqueous chlorine or chlorine dioxide
appeared similar.

141
Product distribution in the TLC fractions. Aliquots of the
radioactive samples (Fig. 7) were spotted on TLC plates and
developed as previously described. The results indicated that
most of the radioactivity was in the polar fractions for both
aqueous chlorine and chlorine dioxide reaction products (Table
20). Due to their high affinity for the silica gel, these
polar compounds had little or no movement from the origin.
The fluorescent compounds in the aqueous chlorine reaction
products accounted for a total of 87.3% of the applied
radioactivity with most of the product (64%) being in the
origin. The chlorine dioxide reaction products accounted for
about 95% of the applied sample and again, most of the
radioactivity was in the polar fractions Bands A and B (92%)
(Table 20).
The highly mutagenic fraction (Band #5) in the aqueous
chlorine reaction was about 1.4% of the total radioactivity,
while in the case of chlorine dioxide, the mutagenic fraction
(Band #C) accounted for only 2% of the total radioactivity
(Table 20).
Mutagenic activity of the TLC extracts of the reaction
products of aqueous chlorine and chlorine dioxide with
tryptophan
The fluorescent bands were scraped from the TLC plates
and the compounds from each band were extracted separately
with acetone. The acetone extracts were then filtered to
remove the silica and concentrated by rotary evaporation under
vacuum. Upon drying, some brown-colored substances similar

142
Table 20. Distribution of radioactivity in the TLC
subfractions of the reaction products of aqueous
chlorine or chlorine dioxide with tryptophan3
Aqueous chlorine (% Radio- Chlorine dioxide (% Radio¬
activity5) activity)
Band # Band #
1 (Rf=0)
64.1
A (Rf=0)
15.0
2 (Rf=0.08 )
11.0
B (Rf=0.13)
77.0
3 (Rf=0.22 )
8.7
C (Rf=0.31)
2.0
4 (Rf=0.38)
1.8
D (Rf=0.44)
0.74
5 (Rf=0.54 )
1.4
Gaps0
3.0
Gaps
0.3
aTryptophan
spiked with
solutions in
radioactive
0.1 M sodium phosphate buffer were
tryptophan of known activity and
allowed to react with aqueous chlorine or chlorine dioxide
solutions at a 7:1 disinfectant:tryptophan ratio for 24 hours.
Any residual chlorine or chlorine dioxide remaining after the
reaction was removed with sodium sulfite. The solutions were
filtered and concentrated by resin adsorption using Amberlite
XAD-2/8. The adsorbed compounds were eluted with acetone and
concentrated. The acetone eluates were then fractionated on
silica gel TLC plates. The different fractions detected by
exposure to UV light were then scraped off and the
radioactivity in the samples was counted by liquid
scintillation counting.
bPercent radioactivity was determined by dividing the
radioactivity count of each band by the total radioactivity
applied to the TLC plates.
cThe silica gel between the fluorescent compounds were also
scraped off and radioactivity determined.

143
to the original material were obtained in all the fractions.
The intensity of the color decreased from the origin to the
least polar product. The dry samples were dissolved in and
diluted with DMSO and assayed for mutagenic activity.
Aqueous chlorine. Mutagenic activity was detected in all the
5 bands of the TLC extracts in strain TA98 without metabolic
activation (Table 21) . From the relative mutagenicity values,
band #4 appeared to be the most potent in this bacteria strain
and closely followed by band #5. Toxicity was observed at 5
mg/plate for bands #2 and #3. When the S-9 mix was added,
there was a sharp decrease in mutagenic activity in all the
samples. Addition of the S-9 mix reduced the mutagenicities
of the extracts and bands 2-4 were only weakly mutagenic while
band #1 was not. Also bands 2 and 3 which showed some
toxicity were no longer toxic (Table 21).
Toward strain TA100, band #1 was barely mutagenic at the
highest dose tested while the rest of the samples exhibited
potent mutagenic activity in the absence of the S-9 mix (Table
22). Bands #2 and 5, both of which showed blue fluorescence,
were the most potent in showing mutagenic activity and were
more so than the non-fractionated extracts. Bands #3 and 4
were also very potent but to a limited degree (Table 22) .
Thus the fractionation process was able to remove some of the
toxic or antimutagenic components in the reaction mixture.
In the presence of the S-9 mix, there was a major reduction
in mutagenic activity such that band #1 tested negative and

144
Table 21. Mutagenicity of the TLC subfractions obtained
from the Amberlite XAD eluates of the reaction
products of aqueous chlorination of tryptophan
using Salmonella tvphimurium strain TA98a
Number of revertants/plateb
TA98
Sample
Dose/plate
-S9
MRC
+S9
MR
Bacteria
27± 2
281 7
Bacteria
+ DMSO
25 mL
22± 2
261 7
Band #1
Rf=0
250
22± 4
1.0*
311 7
1.2*
1 mg
33± 5
1.5
281 2
1.1*
5 mg
78± 6
3.5
301 6
1.2*
Band #2
Rf=0.08
250 Mg
44± 6
2.0
301 7
1.2*
1 mg
174±15
7.9
361 5
1.4*
5 mg
Td
56110
2.2
Band #3
Rf=0.22
250 Mg
2 6± 4
1.2*
281 6
1.1*
1 mg
118±10
5.4
341 5
1.3*
5 mg
T
71119
2.7
Band #4
Rf=0.38
500 Mg
62±10
2.8
261 6
1.0*
1 mg
138± 8
6.3
28110
1.1*
5 mg
598±59
27.2
621 8
2.4
Band #5
Rf=0.54
250 Mg
53± 6
2.4
301 4
1.2*
1 mg
111± 8
5.0
441 5
1.7
5 mg
418120
19.0
891 7
3.4

145
Table 21-continued.
a Aqueous chlorine was allowed to react with tryptophan (7:1
molar ratio) at room temperature for 24 hours. Any excess
chlorine remaining after the reaction was removed with sodium
sulfite and the reaction products were concentrated by
Amberlite XAD adsorption using acetone as eluant. The XAD
eluates were further fractionated by thin layer chromatography
on silica gel G plates developed with a hexane:ethyl
ether:acetic acid (50:50:1, v/v/v) solvent system. The
fractions were then assayed for mutagenic activity in
Salmonella tvphimurium strain TA98.
bResults of duplicate runs of 4 plates/dose. Means and
standard deviations.
cMutagenicity ratio is the number of revertants per test dose
divided by that of the solvent controls. Asterisk denotes not
significantly different from control samples (P=0.05).
dToxic.

146
Table 22. Mutagenicity of the TLC subfractions obtained
from the Amberlite XAD eluates of the reaction
products of aqueous chlorination of tryptophan
using Salmonella tvphimurium strain TA100a
Number of revertants/plateb
TA100
Sample Dose/plate -S9 MRC +S9 MR
Bacteria
12 0±
13
89±
14
Bacteria
+ DMSO
25
/¿L
116±
6
95±
2
Band
#1
Rf=0
250
Mg
113±
4
1.0*
111±
9
1.2
1
mg
158±
15
1.4
110±
14
1.2*
5
mg
321±
30
2.8
124±
20
1.3
Band
#2
Rf=0.08
250
Mg
225±
15
1.9
113±
18
1.2*
1
mg
594±
72
5.1
144±
16
1.7
5
mg
3987±186
34.4
4 3 0±
39
4.5
Band
#3
Rf=0.22
250
Mg
180±
19
1.6
12 6±
9
1.3
1
mg
915±
22
7.9
166±
6
1.7
5
mg
Td
522±
33
5.5
Band
#4
Rf=0.3 8
500
Mg
2 67±
26
2.3
13 0±
15
1.4
1
mg
1420±
88
12.2
154±
11
1.6
5
mg
T
6481113
6.8
Band
#5
Rf=0.54
250
Mg
177±
20
1.5
1461
15
1.2
1
mg
1017±
96
8.8
2131
20
2.2
5
mg
41281419
35.6
7281
47
7.6

147
Table 22-continued.
dAqueous chlorine was allowed to react with tryptophan (7:1
molar ratio) at room temperature for 24 hours. Any excess
chlorine remaining after the reaction was removed with sodium
sulfite and the reaction products were concentrated by
Amberlite XAD adsorption using acetone as eluant. The XAD
eluates were further fractionated by thin layer chromatography
on silica gel G plates developed with a hexane:ethyl
ether:acetic acid (50:50:1, v/v/v) solvent system. The
fractions were then assayed for mutagenic activity in
Salmonella tvphimurium strain TA100.
bResults of duplicate runs of 4 plates/dose. Means and
standard deviations.
cMutagenicity ratio is the number of revertants per test dose
divided by that of the solvent controls. Asterisk denotes not
significantly different from control samples (P=0.05).
dToxic.

148
bands 3 and 4 were mutagenic only at the maximum dose of 5
mg/plate. Also the toxicity in bands 3 and 4 was eliminated.
The most potent mutagenic band (#5) was positive at two doses
but with about a 5-fold decrease in mutagenic activity at the
highest dose tested (Table 22).
For the purposes of the mutagenicity assessment of the
products in band 5 re-developed in the second dimension, the
TLC plates were divided into several portions. All portions
(including both fluorescent and nonfluorescent bands) were
scraped off separately, dissolved in acetone and dried. The
dry samples were then taken up in DMSO and assayed for
mutagenic activity. This assay was performed only in bacteria
strain TA100 without metabolic activation. The results are
presented in Table 23. High mutagenic activity was detected
in only 2 fractions. Band #5b which did not show any
fluorescence was mutagenic at the highest dose tested (Table
23). The only other band which showed higher mutagenic
activity was band #5c (Rf=0.31) which showed blue fluorescence
similar to the original material. This fraction showed a
dose-related increase in mutagenic activity with very high
mutagenicity ratios at the higher doses tested (Table 23).
Chlorine dioxide. Mutagenic activity was detected in all the
TLC bands of chlorine dioxide reaction mixture in strain TA98.
In all cases mutagenic activity was weak and not comparable
to that of the aqueous chlorine samples (Table 24) . As
opposed to aqueous chlorine samples, no toxicities were

149
Table 23. Mutagenicity of the TLC fraction of the reaction
products of the aqueous chlorination of
tryptophan re-developed on a second plate3
Number of revertants/plateb
TA100
Sample
Dose/plate -S9
MRC
Bacteria
109±
11
Bacteria + DMSO
25 /iL
118±
8
Band #
5a (Rf=0)d
1.0 mg
103±
17
0.9*
2.5 mg
141±
13
1.2
5.0 mg
2 31±
18
2.0
5b
(Rf=0.16)
1.0
mg
170±
35
1.4
2.5
mg
216±
16
1.8
5.0
mg
641±
31
5.4
5c
(Rf=0.31)
1.0
mg
150±
7
1.3
2.5
mg
627±
80
5.3
5.0
mg
1520±103
12.9
5d
(Rf=0.46)
1.0
mg
86±
6
0.7*
2.5
mg
117±
7
1.0*
5e
(Rf=0.62)
1.0
mg
93±
11
0.8*
2.5
mg
102±
2
0.9*
4.0
mg
105±
3
0.9*
5f
(Rf=0.65)
1.0
mg
102±
6
0.9*
2.5
mg
115±
2
1.0*
5.0
mg
158±
15
1.3*
5g
(Rf=0.75)
1.0
mg
86±
9
0.7*
2.5
mg
84±
5
0.7*
5.0
mg
104±
8
0.9*
5h
W
II
O
•
CO
CO
1.0
mg
91±
9
0.8*
2.5
mg
95±
5
0.8*
5.0
mg
92±
8
0.8*

150
Table 23-continued
aAqueous chlorine was allowed to react with tryptophan (7:1
molar ratio) at room temperature for 24 hours. Any excess
chlorine remaining after the reaction was removed with sodium
sulfite and the reaction products concentrated by Amberlite
XAD adsorption using acetone as eluant. The XAD eluates were
fractionated by thin layer chromatography on silica gel G
plates developed with hexane:ethyl ether:acetic acid (50:50:1,
v/v/v). A product from the above fraction was further
separated on a second TLC plate using an eluant comprising
hexane:methylene chloride:acetic acid (50:50:2, v/v/v). The
fractions were then assayed for mutagenic activity in
Salmonella tvphimurium strain TA100.
Results of duplicate runs of 4 plates/dose. Means and
standard deviations.
cMutagenicity ratio is the number of revertants per test dose
divided that of the controls. Asterisk denotes not
significantly different from control samples (P=0.05).
dBands 5a, 5b, 5d and 5g did not show fluorescence therefore
their Rf values were taken from the center of the band.

151
Table 24. Mutagenicity of the TLC subfractions obtained
from the Amberlite XAD eluates of the reaction
products of chlorine dioxide and tryptophan using
Salmonella tvohimurium strain TA98a
Number
of revertants/plateb
Sample
Dose/plate
-S9
MR°
+S9
MR
Bacteria
3 0± 7
29± 5
Bacteria
+ DMSO
25 /iL
28± 9
2 6± 7
Band #A
Rf=0
1 mg
47± 6
1.7
2 6± 2
1.0*
2 mg
51± 5
1.8
33± 5
1.3*
5 mg
79±14
2.8
29± 1
1.1*
Band #B
Rf=0.13
1 mg
41± 2
1.5
27± 9
1.0*
2 mg
56± 2
2.0
33± 5
1.3*
5 mg
84± 3
3.2
33± 2
1.3*
Band #C
Rf=0.31
1 mg
NDd
ND
2 mg
83± 4
3.0
33± 4
1.3*
5 mg
132±15
4.7
60± 6
2.3
Band #D
Rf=0.4 4
1 mg
40± 5
1.4*
27± 4
1.0*
2 mg
49± 5
1.8
32± 4
1.2*
a, —-—
5 mg
74± 7
2.6
28± 1
1.1*
(7:1 molar ratio) at room temperature for 24 hours. Any
excess chlorine dioxide remaining after the reaction was
removed with sodium sulfite and reaction products were
concentrated by Amberlite XAD adsorption using acetone as
eluant. The XAD concentrates were further fractionated by
thin layer chromatography using silica gel G plates developed
with a hexane:ethyl ether:methanol:acetic acid (63:30:7:2,
v/v/v/v) solvent system. The fractions were then assayed for
mutagenic activity in Salmonella tvphimurium strain TA98.
Duplicate runs of 4 plates/dose,
deviations.
Means and standard
cMutagenicity ratio is the number of revertants per test dose
divided that of the solvent controls. Asterisk denotes not
significantly different from control samples (P=0.05).
dNot determined.

152
observed in these samples even at the highest dose of 5
mg/plate. Perhaps the strongest mutagen in these extracts
was a narrow blue fluorescent band designated C. In the
presence of the S-9 mix, mutagenic activity was abolished in
all the extracts except band C.
In bacterial strain TA100, band B was weakly mutagenic
at 5 mg/plate and C was mutagenic at 2 doses. Bands A and D
were basically non-mutagenic (Table 25). No observable
changes were observed on addition of the S-9 mix. Again bands
B and C tested positive in the presence of the S-9 mix even
though the activities were weak (Table 25).
Induction of Sister Chromatid Exchange by the TLC
Fractions of the Reaction Products of Aqueous Chlorine
or Chlorine Dioxide with Tryptophan
The mutagenic TLC subfractions of the reaction products
of aqueous chlorine or chlorine dioxide with tryptophan were
further evaluated for genotoxicity using the sister chromatid
exchange assay. For the aqueous chlorine reaction products,
bands 2 through 5 were used while in the case of chlorine
dioxide only bands B and C were used. A high dose of 1 mg/mL,
a low dose of 1 /xg/mL and other intermediate doses were used
for all the treatments. The results indicated that the
reaction products in these bands were capable of significantly
(P=0.05) increasing the sister chromatid exchange frequencies
over the DMSO control (Table 26). Except for sample D, the

153
Table 25. Mutagenicity of the TLC subfractions obtained
from the Amberlite XAD eluates of the reaction
products of chlorine dioxide and tryptophan using
Salmonella tvphimurium strain TA100a
Number
of revertants/plateb
Sample
Dose/plate
-S9
MRL
+S9
MR
Bacteria
106±12
1091 8
Bacteria
+ DMSO
25 mL
98± 7
941 7
Band #A
â– H
II
o
1 mg
129118
1.3
1221 9
1.3
2 mg
118122
1.2*
118110
1.3
5 mg
149118
1.5
1291 9
1.4
Band #B
Rf=0.13
1 mg
128125
1.3*
1221 9
1.3
2 mg
169123
1.7
158116
1.7
5 mg
275123
2.8
214134
2.3
Band #C
Rf=0.31
1 mg
NDd
ND
2 mg
281135
2.9
250113
2.7
5 mg
532165
5.4
489112
5.2
Band #D
o
II
1 mg
103115
1.1*
109110
1.2
2 mg
123114
1.3
106110
1.1*
a„—
5 mg
147115
1.5
128117
1.4
(7:1 molar ratio) at room temperature for 2 4 hours. Any
excess chlorine dioxide remaining after the reaction was
removed with sodium sulfite and reaction products were
concentrated by Amberlite XAD adsorption using acetone as
eluant. The XAD concentrates were further fractionated by
thin layer chromatography using silica gel G plates developed
with a hexane:ethyl ether:methanol:acetic acid (63:30:7:2,
v/v/v/v) solvent system. The extracts were then assayed for
mutagenic activity in Salmonella tvphimurium strain TA100.
duplicate runs of 4 plates/dose,
deviations.
Means and standard
cMutagenicity ratio is the number of revertants per test dose
divided that of the solvent controls. Asterisk denotes not
significantly different from control samples (P=0.05).
dNot determined.

154
Table 26. Sister chromatid exchange frequencies induced by
the TLC subtractions of the reaction products of
aqueous chlorine and chlorine dioxide with
tryptophan3
Sample
Dose
SCE per cellb
DMSO
25 /xL
6.90±2.1
MMS
0.002 /xL
29.15±5.4
Aaueous
chlorine
Band #2,
CO
o
o
II
s-
Pd
200 /xg
11.7512.6
100 /xg
11.16±2.9
10 /xg
7.75±1.9C
Band #3,
Rf=0.2 2
200 /xg
11.3212.7
100 /xg
10.1512.8
10 /xg
6.9412.0C
Band #4,
Rf=0.38
200 /xg
8.7012.6
100 /xg
8.3212.4C
10 /xg
6.9612.2C
Band #5,
Rf=0.54
200 /xg
11.7412.5
100 /xg
9.5011.7
10 /xg
7.1512.2C
Chlorine
dioxide
Band #B,
Rf=0.13
1 mg
10.6012.5
100 /xg
8.1012.5C
10 /xg
6.8912 . lc
Band #C,
Rf=0.31
200 /xg
10.9412.5
100 /xg
9.8512.1
a,
10 /xg
6.8411.7C
with tryptophan (7:1 molar ratio) for 24 hours. The
reaction mixture was concentrated by resin adsorption and
the adsorbed compounds were eluted with acetone. The
acetone eluates were further fractionated by thin layer
chromatography on silica gel G plates. Compounds in the TLC
fractions were extracted with acetone. Following proper
processing, assayed for genotoxicity using the sister
chromatid exchange assay in CHO cells.
bThe data were tranformed logarithmically and analyzed for
statistical significance using the Dunnett's test.
cNot significantly different from DMSO control sample.

155
highest doses tested (1 mg/itiL) did not provide enough
metaphases for chromosomal analysis. Bands 2 and 5 were the
most genotoxic of the aqueous chlorine reaction products at
the highest positive doses. The other aqueous chlorine and
chlorine dioxide fractions also showed genotoxic activity.
This is in line with the Ames mutagenicity data. Statistical
analysis of the results using the Dunnett's test (Steel and
Torrie, 1980) showed that all the samples were significantly
different (P=0.05) from the control samples at the highest
non-toxic doses tested. A test dose of 10 ¿¿g/mL was found to
be non significantly different in all the treatments. Band
#4 and #C were also not significantly different (P=0.05) from
control samples (Table 26).
Identification of the Reaction Products
For the aqueous chlorine reaction products, the neutral
methylene chloride and hexane extracts from the preliminary
liquid-liquid extraction study as well as the highly mutagenic
TLC subfraction (band #5) were analyzed by GC/MS. The
methylene chloride and hexane extracts contained several
compounds; some of which have previously been identified by
other researchers. A reconstructed ion chromatogram of the
products from the methylene extracts is presented in Fig. 8.
Three indole derivatives, oxindole, isatin and indolyl-
acetonitrile, and two quinoline-type compounds, norhaman and
3-hydroxyquinaldonitrile, were tentatively identified from

RIC
156
Scan Number
Figure 8. Reconstructed ion chromatogram of the methylene
chloride extract of the reaction products of
aqueous chlorine with tryptophan. 1) oxindole
2) indolylacetonitrile 3) isatin, and
4) norhaman.

157
this extract. The precipitate was analyzed by direct
insertion probe and heated on the column but was found to be
highly nonvolatile with the bulk of the materials coming up
late in the program as combustion products. The only
chlorinated compounds detected in this fraction resembled
those of trisubstituted chlorophenols which probably arose
from the water. Chlorophenols are used as wood preservatives
and it is not uncommon to find them in water.
The mass chromatogram of the products from the TLC
subfraction (band #5) is presented in Fig. 9. Three
chlorinated compounds were present in this fraction. Two
chloroketones, 1,1,3-trichloropropanone and 1,1,3,3-tetra-
chloropropanone and one guinoline compound, dichloroquinoline
were tentatively identified in this fraction. Another TLC
subfraction (band #4) was analyzed (Appendix) and found to
contain four chlorinated compounds. Some of the compounds
such as chloral have been identified in chlorinated humic acid
solutions (Horth et al., 1987). Another compound similar to
dichloroguinoline but with three chlorines attached was
present in this fraction (Appendix).
The mass chromatogram of the weakly mutagenic TLC
subfraction of the chlorine dioxide reaction products was a
simple one containing only two compounds (Fig. 10) . Computer
analysis of the spectra of the compounds from this fraction
with compounds in its library did find any close matches. The

158
spectra
pentanol
for these compounds resembled those 2
and 2,4-dimethyl pentane.
,4-dimethyl

RIC
159
Scan Number
Figure 9. Reconstructed ion chromatogram of the of the thin
layer chromatography subfraction of the reaction
products of chlorine with tryptophan (band 5).
1) 1,1,3-trichloropropanone 2) 1,1,3,3-tetra-
chloropropanone, and 3) dichloroquinoline.

Scan Number
Figure 10. Reconstructed ion chromatogram of the thin layer
chromatography subfraction of the reaction
products of chlorine dioxide with tryptophan
(band C).

CHAPTER V
DISCUSSION
Due to its efficiency, low cost, ease of application and
adequate persistence in water, chlorine has been widely used
since World War II for water disinfection (White, 1972).
Because of its continued use, several studies have been
conducted by different researchers in order to understand the
chemistry and fate of chlorine in drinking water. As a result
of these studies, researchers have gained a lot of information
on the use and possible health risks posed by chlorine in
drinking water treatment. Also the reactions of chlorine with
amino acids in dilute aqueous solutions have been studied in
some detail. The early studies on the chlorination of amino
acids focused mainly on the identification of the reaction
products (Dakin et al., 1916; Langheld, 1909), while more
recent studies have concentrated on the genotoxic properties
as well as identification of the mutagenic reaction products
likely to be produced from such reactions (Horth et al., 1987;
Trehy et al., 1986).
161

162
Reactions of Aqueous Chlorine and Chlorine Dioxide with
Tryptophan
Tryptophan has been used as a model in different studies
to investigate chloroform formation or mutagenic activity in
aqueous chlorine solutions (Sussmuth, 1982; Morris and Baum,
1978). Kirk and Mitchell (1980) investigated the reactions
of aqueous chlorine and tryptophan using different
concentrations and observed the formation of a dark-colored
precipitate upon reaction. Burleson et al. (1980) also
observed a reddish color formation in their ether extracts.
In the present study, a dark-colored reaction mixture
and precipitate were formed upon mixing tryptophan and aqueous
chlorine solutions at three different molar ratios (chlorine:
tryptophan, 1:1, 3:1 and 7:1). The precipitate continued to
be formed even when the reaction products were concentrated
by Amberlite XAD-2/8 adsorption and kept refrigerated. When
the molar concentration of chlorine was increased more than
15-fold over that of the amino acid, color formation was not
observed. Other researchers have studied similar reactions
in model systems but did not report color formation (Horth et
al., 1987; Trehy et al., 1986; Sussmuth, 1982). This is
probably due to the fact that the amount of chlorine used in
these studies was in excess of the amino acid which precluded
color formation. Sussmuth (1982) bubbled 80 mL of chlorine
gas into a solution containing 250 mg of tryptophan; Trehy and
coworkers (1986) used a chlorine to amino acid ratio of 14.

163
At these ratios, color and precipitate formation were either
not observed or not reported.
In the case of chlorine dioxide, very little information
is available in the literature regarding its reactions with
amino acids. Masschelein (1979) reported that tryptophan was
very reactive with chlorine dioxide leading to the formation
of reddish coloration. In this study, reddish coloration was
observed when excess chlorine dioxide was reacted with
tryptophan. The reddish color changed to a dark color when
the reaction progressed. The amount of precipitate and
intensity of color formed from the chlorine dioxide reaction
mixtures were much less than that of aqueous chlorine. The
only other report in the literature on the reaction of
chlorine dioxide with tryptophan was published in 1957 by
Fujii and Ukita who reported formation of complex reaction
mixtures containing indigo red as well as unidentified
polymerization products. Structurally similar indole
compounds have been shown to react with chlorine dioxide under
water treatment conditions (Lin and Carlson, 1984). Chlorine
dioxide oxidation of indole produces oxindole and isatin as
major products, while with 3-methylindole, 3-methyloxindole
and o-formamidoacetophenone were produced as the major
products. Color formation in the reactions between chlorine
dioxide and tyrosine has been well documented and is used as
a basis for the determination of chlorine dioxide
concentrations in water (Hogden and Ingols, 1954).

164
In general methyl ketones or compounds oxidizable to
these structures undergo base-catalyzed halogenation reactions
leading to the formation of chloroform and other THM (Rook,
1974). The reactivity of tryptophan and structurally similar
pyrrole compounds is due to the fact they are capable of
active carbanion formation. Due to their reactive nature,
such compounds are capable of rapid reaction with chlorine
leading to formation of chloroform and other THM in a
mechanism similar to the haloform reaction (Morris and Baum,
1978). According to Morris and Baum (1978), acetone or other
simple methyl ketones react too slowly to account for
chloroform formation underwater chlorination conditions. For
acetone, the computed half-life for haloform formation at
neutral pH and at ambient temperature is almost one year.
Compounds such as beta diketones and the pyrroles are capable
of giving chloroform yields approaching one mole per mole of
compound within a few hours (Morris and Baum, 1978). In model
chlorination studies, Morris and Baum (1978) observed linear
increases in chloroform formation when excess chlorine was
reacted with tryptophan. The molar yields of chloroform in
these studies were about 18% at pH 7.5 compared to about 100%
at pH 11.0. Tryptophan reacted with chlorine under neutral
or slightly acidic pH conditions with chloroform being
liberated at elevated pH. Thus, tryptophan and structurally
similar pyrrole compounds may account for some of the haloform
formation under water chlorination conditions.

165
Mutagenicity Evaluation
Short-term in vitro bioassays have become very popular
over the past decade or so primarily because they can be
performed relatively easily and inexpensively compared to
long-term carcinogenicity assays which often involve the use
of several rodents and other mammals. Also, protests from
animal groups have led to serious considerations about using
alternate in vitro assays which do not require the use of test
animals. The best known and perhaps most widely used
short-term assay for detecting mutagenic and potentially
carcinogenic compounds in the environment and industry is the
Ames Salmonella/mammalian microsome assay. This is because
it is relatively easy to perform and has a sizeable literature
base (Tennant et al., 1987). The Ames test uses different
strains of S. tvphimurium bacteria which have been genetically
modified to detect mutagens and potential carcinogens. The
most sensitive and widely used bacteria, strains TA98 and
TA100 are used to detect frameshift and base-pair substitution
mutagens, respectively. The tests can also be performed with
the addition of mammalian liver homogenate (rat liver S-9
fraction) which contains all essential drug metabolizing
enzymes to simulate mammalian metabolism. Other recognized
in vitro short-term assays include chromosomal aberrations,
sister chromatid exchange and mouse lymphoma cells. Even
though these assays have their inherent limitations (e.g.

166
false positives and negatives), they are nonetheless used
extensively for screening chemical carcinogens and mutagens.
The original report of the Ames test which indicated a
90% correlation between carcinogenicity and mutagenicity
(McCann et al., 1975), was later revised to about 83% by the
same authors (Ames and McCann, 1981). More recently, the
correlation has been shown to be much less than originally
predicted. Tennant et al. (1987) have reported that test
results from four in vitro short-term assays including the
Ames test did not show concordance with rodent carcinogenicity
data and that concordance of each assay was about 60%. There
was, however, good agreement between the short-term tests in
that when a chemical tested positive in one assay, it tended
to be positive in the other three assays. As pointed out by
the authors, short-term tests have been used successfully in
certain areas where whole animal assays have not been used.
For example short-term tests have been used for the analysis
of complex mixtures of cooked meat, chlorinated water, air
pollutants and for the early detection and identification of
genotoxicants before manufacture. In summary, even though
short-term tests are useful in their own way, they cannot
replace whole animal assays completely.
Concentration of the reaction products
As stated earlier, the versatility of the Ames and other
short-term tests lies in their ability to detect chemical
carcinogens and/or mutagens in complex environmental mixtures.

167
Except for a few compounds e.g. chloroform, chemical mutagens
are usually formed at the microgram per liter (ppb), or lower
levels in chlorinated drinking water (Tardiff et al., 1978).
It is therefore necessary to concentrate drinking water
samples by several orders of magnitude for chemical analysis
and/or genotoxicity assessment. Some researchers have
reported mutagenic activity in unconcentrated water samples
but the reported activities are generally weak (Kool et al.,
1981a).
Several methods are available for achieving this
concentration step prior to analysis. These include
liquid-liquid extraction, freeze drying, reverse osmosis and
resin or activated carbon adsorption/desorption. Method
selection for sample concentration has been found greatly to
influence the results of different mutagenicity data, and as
such, values reported from different sources may not be
directly comparable (Vartiainen et al., 1987). Of the
sorption methods, those that have been very well documented
are the Amberlite resins XAD-2, XAD-4, XAD-7 and XAD-8
(Daignault et al., 1988). In this study, rotary evaporation,
liquid-liquid extraction and Amberlite XAD polymeric resin
adsorption were used to concentrate the reaction mixtures for
genotoxicity assessment.
Rotary evaporation
This method is generally not employed in water
chlorination studies because these studies require large

168
volumes of water (50 to 100 L) (Monarca et al., 1985; Grabow
et al.; 1981). Also, the method is tedious and time consuming
and most of the volatile materials may be lost if samples are
concentrated this way. In the present study, mutagenic
activity was detected in the complex reaction mixtures
containing equimolar concentrations of aqueous chlorine and
tryptophan concentrated by rotary evaporation. Mutagenic
activity was decreased in all the samples in the presence of
the rat liver enzyme system. The binding of the S-9 mix to
the sample may have decreased the quantity of test chemicals
available for bacterial mutagenesis, or the production of less
toxic metabolites via the mixed function oxidase enzyme system
may have led to a decline in the mutagenic activity. These
results are in agreement with previously reported data
(Sussmuth, 1982). Also at high doses (300 /¿L) the number of
revertants was decreased indicating that the chemicals were
causing some toxicity to the bacteria. There is a
million-fold range of differences in carcinogenic and
mutagenic potency among various chemicals tested in female
mice and the Ames mutagenicity assay, respectively (McCann,
1983). Thus, some compounds require much higher
concentrations to show the same carcinogenic and/or mutagenic
effects as others.
The compounds were mainly direct-acting, nonvolatile
mutagens. Nonvolatile materials which are also direct-acting
have been shown to be produced as a result of water

169
chlorination and account for a substantial portion of the
mutagenic activity observed in drinking water concentrates
(Grabow et al., 1981; Cheh et al., 1980). Direct-acting and
promutagens have been detected in unconcentrated water from
the lower Mississippi river which include both volatile and
nonvolatile materials (Pelón et al., 1977). Volatile
materials such as chloroform and other THM are also produced
as a result of chlorination and may contribute to the overall
mutagenic activity in drinking water, but for the purposes of
this study they are not expected to account for any of the
observed mutagenic activity.
Liquid-liquid extraction
Liquid-liquid extraction by mechanical mixing, for
example by magnetic stirring, has been used by several
researchers to concentrate organic compounds from aqueous
solutions for mutagenicity assessment and/or chemical analysis
(Vartiainen et al., 1987; Trehy et al., 1986; Grabow, 1981).
Other methods include continuous liquid-liquid extraction or
Ultra-Turrax mixing (Vartiainen et al., 1987). The results
from these studies differ slightly regarding solvent selection
and extraction time. Traditionally, organic solvents like
methylene chloride, ethyl ether, pentane, hexane and ethyl
acetate or a combination of these or other solvents immiscible
with water are used for these extractions.

170
Type of solvent and extraction time. In preliminary studies,
liquid-liquid extractions were employed usinq hexane, ethyl
ether and dichloromethane to extract the reaction products
from equimolar concentrations of aqueous chlorine-tryptophan
mixtures after sample pH adjustment. The results indicated
that ethyl ether was more suitable than either dichloromethane
or hexane (Tables 4 and 5) . These results are in agreement
with other published data (Vartiainen et al., 1987; Trehy et
al., 1986). Even though both hexane and dichloromethane were
capable of extracting some mutagens, the overall mutagenic
activity in these extracts was lower than the ethyl ether
extracts. Higher concentrations of the reaction products were
found to be highly toxic to the bacteria, especially the
neutral ethyl ether and dichloromethane extracts (Tables 4 and
5). Similar toxicities have been observed in drinking water
concentrates (Grabow et al., 1980).
The use of some organic solvents has inherent
disadvantages in that some of them, for example
dichloromethane, have been shown to be carcinogenic and/or
mutagenic. Dichloromethane has been found to be mutagenic in
the Ames test when assayed in an open container inside a
desiccator, but nonmutagenic when incorporated into the agar
(Nestmann et al., 1980) . Dichloromethane was found to be non¬
mutagenic when assayed in the HGPRT forward mutation assay in
CHO cells. However, it caused a slight increase in SCE
frequencies in V9 cells (Nestmann et al., 1981). It has also

171
been reported to be carcinogenic but not teratogenic in male
and female rats (NTP, 1982) . The dichloromethane used in the
present assay is not expected to contribute to any of the
observed mutagenic activity since the samples were poured
directly onto the agar plates and control samples of the
dichloromethane solvent alone were non-mutagenic.
Another concern about the use of some organic solvents
in sample concentration by liquid-liquid extraction is the
possibility of the formation of toxic metabolites from the
reaction of residual chlorine with the solvent.
Vartiainen and coworkers (1987) used liquid-liquid
extraction either with magnetic stirring or Ultra-Turrax
mixing to concentrate Finnish drinking water. They reported
that the concentrates obtained were mutagenic only in the
presence of salt in the extraction procedure. Other
researchers have used liquid-liquid extraction by Ultra-Turrax
mixing without addition of salt and detected mutagenic
activity in their extracts (Grabow et al., 1981). Vartiainen
and colleagues (1987) also reported that continuous liquid-
liquid extraction with ethyl ether and 20% salt was more
efficient than using dichloromethane under similar conditions.
In their studies, increased mutagenic activity was observed
with longer extraction times (up to 48 hours). In the present
study, no salt was added and only mechanical mixing with a
magnetic stirrer was used. In the reaction mixtures with
aqueous chlorine, higher mutagenic activity was observed at

172
the 1:1 ratio than at the 3:1 ratio. However, mutagenic
activity was highest at the 7:1 ratio (Table 8). The decrease
in activity at the 3:1 ratio may be due to the formation of
less mutagenic products at this ratio. Similar results were
observed for the three ratios of chlorine dioxide and
tryptophan. However, the differences between the 1:1 and 3:1
ratios were not specific (Table 10). At high concentrations
(5 mg/plate), toxicity was detected in the aqueous chlorine
but not chlorine dioxide extracts. Bacteria strain TA98 was
found to be more sensitive than TA100 indicating that the
ethyl ether extracts were mainly frameshift mutagens even
though some base-pair substitution mutations were also
detected (Tables 8-11). Salt is usually added to liquid-
liquid extracts to aid in the separation of phases and thereby
increase recovery. It is not known whether addition of salt
would have increased the efficiency of the solvent extractions
and thereby the mutagenic activity.
Vartiainen et al. (1987) have reported that magnetic
stirring for 15 minutes recovered only 5% of the mutagenic
activity in Finnish water while stirring for 4 hours increased
mutagen recovery to 25%. According to the authors, the most
straightforward method of magnetic stirring did not give good
results. However, this was not the case with our samples
since the mutagenicity results from the liquid-liquid
extractions correlated well with those of the XAD resin
adsorption. Trehy et al. (1986) reported that 15 minutes

173
extraction time was adequate for the identification of
components of interest from aqueous chlorine-tryptophan
reaction mixtures. The authors also reported that ethyl ether
was more suitable for solvent extraction of chlorination
reaction products than pentane.
Effect of pH. pH is an important parameter to be considered
in the separation of different classes of compounds by their
acid/base behavior in drinking water prior to toxicological
evaluation or chemical analysis. The order of the pH
adjustment steps is essential since most of the mutagenic
activity of the acid/neutral fraction is destroyed at high
(alkaline) pH (Vartiainen et al., 1987). In this study, the
neutral compounds were extracted before the acid or alkaline
fractions which were also extracted separately (Fig. 5). The
sequence of extraction which is similar to other published
reports (Grabow et al., 1981), was expected to prevent the
destruction of acidic compounds under alkaline conditions.
Most of the mutagenic activity was detected in the neutral and
acidic fractions (Tables 4 and 5) . Some mutagenic activity
was also detected in the alkaline fraction but was relatively
weak. These findings are in agreement with other water
chlorination results (Grabow et al., 1980).
In general, most of the observed mutagenic activity in
drinking water samples have been found in the neutral fraction
(Kool et al., 1982 ; Maruoka and Yamanaka, 1982). This is
important from the standpoint of safety because water destined

174
for human consumption is at or around a pH of neutrality.
Mutagenic activity is also associated with acidic compounds
as well (Monarca et al., 1985; Holmbolm et al., 1984). Basic
compounds usually account for very little or no mutagenic
activity in drinking water. According to Maruoka and Yamanaka
(1982), even though the level of extractable basic compounds
in water is low (about 5%), these may probably contain some
of the strongest mutagens.
Amberlite XAD resin adsorption
Amberlite XAD polymeric resins have received much
attention in recent years because they have been used to
concentrate minute quantities of organic compounds from large
volumes of water. They have also been used for the
concentration of mutagens and carcinogens from human urine
(Yamasaki and Ames, 1977) . Amberlite XAD resins are used
widely because the method is rapid, easy to perform and
requires much less organic solvent than liquid-liquid
extractions. The resins are produced by Rohm and Haas
(Philadelphia, PA) in different forms and polarities and with
different physical properties. Amberlite XAD-2 is a non-polar
polystyrene divinylbenzene copolymer, while XAD-8 is a methyl
methacrylate polymer of intermediate polarity (Rohm and Haas,
1978 Technical Bulletin). Some of the physical properties of
these resins are listed in Table 27.
Different researchers have studied mutagen formation in
drinking water and model reactions using Amberlite XAD resin

175
Table 27. Some physical properties of Amberlite XAD-2 and
XAD-8 resins3
Resin
Chemical
Surface
Polarity
Average pore
Mesh
nature
area
diameter
size
(m2/g)
(Angstrom)
XAD-2
Polystyrene
330
Nonpolar
90
20-50
XAD-8
Acrylic ester 140
Intermediate 250
25-50
aRohm and Haas Technical Bulletin, 1978.

176
concentration. Amberlite XAD-2 resins have been used alone
(Horth et al., 1987; Tan et al., 1987b), or in combination
with Amberlite XAD-8 (Meier et al., 1987; Ringhand et al.,
1987). Amberlite XAD-4 resins have also been used alone
(Vartiainen et al., 1987) or in combination with XAD-8 (Kool
et al., 1981b). Other Amberlite resins such as XAD-7 have
been used with varying success (Grabow et al., 1981). In the
present study, the use of Amberlite XAD-2 and XAD-8 (1:1
mixture) in a single column was found to be more efficient
(45-48% recovery) than using either resin alone (38% recovery)
(Table 12).
The results of the Amberlite XAD resin adsorption study
indicated that XAD-2/8 resins (1:1 mixture) were effective in
concentrating mutagens in our model reaction mixtures of
aqueous chlorine and chlorine dioxide with tryptophan. Both
types of chlorination showed linear dose-related increases in
mutagenic activity. With aqueous chlorine, the mutagenicity
results confirm other studies in which Amberlite XAD-2
concentrating procedures were used (Horth et al., 1987; Tan
et al., 1987b). However, this is the first report to show
that increasing the ratio of aqueous chlorine over tryptophan,
leads to an increase in mutagenic activity (Table 16). Under
the conditions of this study, the highest mutagenic activity
was detected in the 7:1 chlorine to tryptophan ratio (Table
16) . In the case of chlorine dioxide, we have previously
shown that its reaction with tryptophan (1:1 molar ratio)

177
leads to mutagen formation in the Ames test (Sen et al.,
1989). The present study confirms our previous results that
chlorine dioxide and tryptophan reaction mixtures (1:1 molar
ratio) are mutagenic. Also, this study has shown that
increasing the concentration of chlorine dioxide leads to
increased mutagenic activity. These are the only studies
showing that the reaction products of chlorine dioxide with
tryptophan are mutagenic. Like other chlorination studies,
the addition of the S-9 mix led to a decrease in the mutagenic
activity. The only other report in the literature regarding
mutagenicity studies of chlorine dioxide-tryptophan reaction
products was by Tan et al. (1987b). Their results indicated
that the reaction products of chlorine dioxide with tryptophan
were not mutagenic at a 1:1 molar ratio. The discrepancies
observed in this study and Tan's work may be due to the fact
that Tan et al. used only Amberlite XAD-2 for concentrating
their reaction products at pH 6.0 with ethyl ether elution.
As a result, they recovered less than 12% of the radioactivity
applied to their column. In this study, the use of both
Amberlite XAD-2 and XAD-8 in one column with sample pH
adjustment to pH 2.5 allowed for better recovery (about 60%)
of the reaction products. Also, acetone, methanol and ethyl
ether were used as eluants even though they were analyzed
separately for mutagenic activity.
Type of eluant. Different eluants such as methanol, ethyl
ether, acetone, ethyl acetate and DMSO are used to desorb

178
organic compounds from XAD resins. Of these, ethyl ether is
perhaps the most widely used (Daignault et al., 1988). Due
to its relatively nonpolar nature, ethyl ether allows for some
polar interactions to occur via the oxygen and has found many
applications for desorbing relatively nonpolar compounds from
resin columns. It, however, has some disadvantages including
flammability and production of side products such as
2-epoxypropane, hexane, hex-l-ene and 2-methyl pentane (Scott
et al., 1984). Even though it may be suitable for desorbing
some classes of compounds, ethyl ether is not suitable for
others. Tan et al. (1987b) recovered less than 12% of their
chlorination reaction products from XAD-2 columns, while Kool
et al. (1981a) were able to recover only about 20% of the
mutagenic activity from their XAD-4/8 resin column with ethyl
ether. Subsequent elution with either DMSO or acetone yielded
the remaining mutagenic activity (Kool et al., 1981a).
Artifact formation. Amberlite XAD-4 resins have been reported
to produce more artifacts than XAD-2 or XAD-8 resins when used
for concentrating chlorination reaction mixtures, especially
when free chlorine is present in the samples (Sweeney and
Cheh, 1985; Hunt and Pangaro, 1982). Generally, artifacts,
which include naphthalenes, alkyl benzenes, phthalate esters,
styrenes, and a host of other compounds have been associated
with the resin manufacturing process (Daignault et al., 1988;
James et al., 1981). Amberlite XAD-4 has been reported to
produce more artifacts because of its physical

179
characteristics. With a high porosity and surface area (750
m2/g), XAD-4 resins are believed to trap more artifacts than
either XAD-2 (330 m2/g) or XAD-8 (140 m2/g) during manufacture
(Daignault et al., 1988). Different theories have been put
forward to explain artifact release from resin beads, all of
which involve release of artifacts from the interstitial
spaces deep within the beads (Daignault et al., 1988).
Artifacts are thought to arise due to resin drying therefore
resins are stored in a solvent, usually methanol (Junk et al.,
1974). Sudden changes in solvent between aqueous-organic and
back to aqueous have been found to correlate with artifact
release. A study by Scott et al. (1984) showed that
interaction of methanol and water with resin resulted in heat
production with subsequent bead rupture and artifact release.
It has been reported that part of the mutagenicity in
drinking water concentrated by Amberlite XAD adsorption may
be actually due to artifacts from the resins or by reaction
of chlorine with the resin (Daignault et al., 1988). In the
present study, Amberlite XAD-2 and XAD-8 which have been
reported to yield fewer artifacts, were used. Procedural
blanks containing aqueous chlorine or chlorine dioxide alone
in sodium phosphate buffer were run through the resins after
removing any residual chlorine or chlorine dioxide with sodium
sulfite. Also tryptophan blanks in phosphate buffer were
passed through the XAD-2/8 resins to determine if artifacts
from the resins contributed to mutagen formation. None of the

180
procedural blanks was found to be mutagenic (Table 15) . These
results and those of the liquid-liquid extractions confirm
that mutagenic activity in our samples was not due to artifact
formation. Studies by other workers have shown that distilled
water blanks did not produce mutagens in XAD-2 or XAD-7 resins
(Loper et al., 1983; Williams et al., 1982).
The effect of pH. pH is an important parameter which has been
shown to affect the degree and efficiency of resin adsorption
(Ringhand et al., 1987, Jolley, 1981). Because Amberlite XAD
resins are nonionic, ionic organic compounds are adsorbed more
efficiently on the beads at a pH at which ionization is
suppressed. Neutral compounds on the other hand, are adsorbed
irrespective of pH (Ringhand et al., 1987). Also, acidic and
basic compounds can be adsorbed and desorbed very efficiently
with simple pH manipulation (Daignault et al., 1988) . In this
study, pH adjustment of the reaction mixture to pH 2.5 prior
to Amberlite XAD adsorption was found to increase product
recovery when compared to adsorption at neutral pH (Table 13).
This indicates that mutagenic activity in the reaction
mixtures is mainly due to acidic/neutral compounds. These
results confirm other water chlorination results in which more
mutagens were observed under conditions of acidic than
slightly basic or alkaline pH (Ringhand et al., 1987).
Effect of dechlorinating agents. Several agents such as
sodium sulfite, sulfur dioxide, sodium thiosulfate and other
sulfur compounds have been used to dechlorinate water samples

181
prior to sample concentration for mutagenicity evaluation.
Dechlorinating agents are used to remove excess chlorine in
order to prevent any reaction of chlorine with the
concentrating agent such as organic solvents or Amberlite XAD
resins. They are also used to stop the chlorination reaction.
The use of these agents for chlorine removal has been reported
to reduce the mutagenic activity of drinking water
concentrates (Wilcox and Denny, 1985; Cheh et al., 1980).
Cheh and colleagues reported that total removal of chlorine
with sulfite caused a substantial reduction (50-80%) in the
mutagenic activity of their XAD-4 resin concentrates of
drinking water. Partial dechlorination (0.5 mg/L residual)
has been reported to have very little or no effect on the
mutagenic activity in drinking water concentrates (Wilcox and
Denny, 1985); therefore, several researchers are using this
approach to study mutagen formation in drinking water (Horth
et al., 1987) .
In water treatment plants, partial dechlorination is used
to remove part of the applied chlorine in order to leave a
residual for continued disinfection throughout the
distribution system (Wilcox and Denny, 1985). Partial
dechlorination in the final stage of water treatment also
reduces the taste of chlorine in drinking water and makes it
acceptable to the consumer. This practice will likely have
no effect on the mutagenic activity in finished drinking water
since the level of mutagens will still be high. In order to

182
circumvent the dechlorination problem, some researchers use
longer reaction times to allow excess chlorine to evaporate,
or bubble nitrogen gas through the mixture to facilitate
excess chlorine removal (Ringhand et al., 1987; Vartiainen et
al., 1987; Rapson et al., 1985).
In the present study, reaction of aqueous chlorine or
chlorine dioxide with tryptophan at the 7:1 ratio for 24 hours
usually left very little chlorine residual which was
dechlorinated with sodium sulfite. Whether the sulfite was
responsible for reducing some of the mutagenic activity in
our samples is not known since mutagenic activity was highest
in those dechlorinated samples, i.e., the 7:1 aqueous chlorine
or chlorine dioxide:tryptophan reaction mixtures.
Fractionation of the reaction products
In drinking water chlorination studies, several
difficulties arise in sample fractionation for mutagenicity
assessment due to the diverse and complex nature of the
chemicals found in drinking water. As a result, very few
researchers have attempted further product fractionation for
mutagenicity studies. In most cases, the amount of recovered
mutagenicity does not equal the total mutagenic activity in
the samples prior to the fractionation step. This is probably
due to the fact that since organic mutagens are formed in very
minute quantities, some small losses will severely impair the
results. Several chromatographic techniques are available for

183
sample fractionation or separation of which HPLC is the most
popular.
TLC separation. Good resolution of the reaction products (XAD
eluates) was achieved by using TLC separation on silica gel
plates. This mode of separation is equivalent to normal phase
HPLC separation and the advantage of the TLC is that for
mutagenicity evaluation, there is no dilution of the sample
by the mobile phase. In this study, five fluorescent
compounds or mixtures of compounds were obtained under UV
light (366 nm) when excess aqueous chlorine was reacted with
tryptophan (7:1 molar ratio). Development of band #5 of the
aqueous chlorine extract on a second TLC plate revealed that
the fluorescent bands were composed of a mixture of compounds
(Table 23) . Also, excess chlorine dioxide reacted with
tryptophan to obtain four fluorescent bands which were also
probably composed of a mixture. The fluorescent compounds
were probably photosensitive because some loss of fluorescence
was observed when the compounds were exposed to light. Also,
leaving the plates in the dark for an extended period of time
(about 48 hours) led to about 90% loss of fluorescence. This
is the first report in which the reactions of aqueous chlorine
and chlorine dioxide have been shown to produce fluorescent
compounds with tryptophan. Other fluorescent compounds have
been reported to be produced when tryptophan is treated with
sodium nitrite under acidic conditions (Ohara et al., 1988).

184
Tryptophan has a UV absorbance at 280 nm which is used
spectrophotometrically to determine protein concentrations in
protein assays. Reactions of tryptophan with these chlorine
compounds led to a strong UV absorbance in the long wavelength
range (366 nm) and a weak absorbance in the short wavelength
range (254 nm).
The TLC subtractions of the aqueous chlorine and
tryptophan reaction products at the 7:1 ratio, gave the
strongest mutagens. The least polar fraction band #5 which
showed blue fluorescence was the most mutagenic. Other
relatively polar compounds showing green fluorescence (bands
#3 and #4) were also mutagenic (Tables 21 and 22). Further
separation of the products from the highly mutagenic band #5
on another TLC plate produced one fraction which was highly
mutagenic. The mutagenic activity in the latter which also
showed blue fluorescence was about 3 3% of that of band #5
(Table 23). The difference between the TLC fractions and the
original material, i.e. XAD and liquid-liquid extraction
samples, is that the original samples were mainly frameshift
mutagens (TA98) while the TLC fractions were mainly base-pair
substitution mutagens (TA100) (Tables 16 and 21) .
Determination of product distribution in the TLC fractions
using radioactivity indicated that most of the product (about
80%) was in the polar fraction (bands #1 and 2). The highly
mutagenic fraction (band #5) accounted for less than 2% of the
total radioactivity in the TLC fractions (Table 20). Further

185
fractionation of this band led the isolation of a potent
mutagenic band which is expected to account for no more than
1% of the total mutagenicity in the reaction products. These
results were expected since organic mutagens are usually
formed at very low levels (/ig/L or ppb) in drinking water.
In the case of the chlorine dioxide reaction products,
weak mutagenic activity was detected in one polar (band B)
and another slightly polar fraction (band C). Again, most of
the radioactivity (93%) was in the polar fractions (bands A
and B) (Table 20) . Mutagenicity in these extracts was also
mainly base-pair substitution mutagens which were relatively
weak (Tables 24 and 25). The only fraction in these samples
which can be considered mutagenic (band C) represented only
about 2% of the applied material (Table 20). This mutagenic
fraction was not deemed potent enough to warrant further
fractionation.
Comparison of licruid-1 icruid extraction and Amberlite
XAD-2/8 adsorption
This study was designed such that liquid-liquid
extraction and Amberlite XAD resin adsorption could be
compared directly. The reaction mixtures, usually 2 liters,
were split into two portions with one-half for resin
adsorption and the other half for liquid-liquid extraction
(Fig. 6) . The different doses used for each mutagenicity
assay preclude any direct comparison. However, based on the
raw data, it is evident that Amberlite XAD-2/8 resin

186
adsorption was more efficient than liquid-liquid extraction
of the aqueous chlorine-tryptophan reaction mixtures (Fig.
11a) . In this figure, the amount of sample used in the
mutagenicity assay as processed for the liquid-liquid
extraction was equivalent to 1 mg dry extract/plate, while
the amount used as prepared by acetone elution of the XAD
resins was equivalent to 100 /iL/plate, or 0.1 mg/plate. On
a weight-to-weight basis, the liquid-liquid extracts were
about ten-fold more concentrated than the resin eluates.
Statistical analysis of the data using the Student's t-test
and comparing the individual ratios i.e. 1:1 resin versus 1:1
liquid-liquid extraction indicated that the resins
concentrated significantly (P<0.05) more mutagens than the
liquid-liquid extractions at all the levels tested (Fig. 11a) .
At the 1:1 aqueous chlorine to tryptophan ratio, liquid-liquid
extraction produced about 49% the number of revertants in the
XAD eluates, while at the 3:1 ratio, the number was less
(about 28%) . When the ratio of chlorine was increased further
to 7:1, the total number of revertants in the liquid-liquid
extracts increased to about 7 6% of that of the acetone eluates
from the resins. Similar results were obtained for the
chlorine dioxide-tryptophan reactions (Fig. lib).
Two studies in the literature have compared liquid-liquid
extraction and Amberlite XAD resin adsorption for
concentrating mutagens in drinking water. Grabow et al.
(1981) reported that liquid-liquid extraction by Ultra-Turrax

Number of revertants Number of revertants
187
Chlorine to tryptophan ratio
Figure 11. Comparison of liquid-liquid extraction (LLE, dose
1 mg/plate) and Amberlite XAD 2/8 adsorption
(XAD, dose 100 /¿L/plate) . A) aqueous chlorine
and B) chlorine dioxide.

188
homogenization for 5 minutes using dichloromethane was
superior to Amberlite XAD adsorption. Vartiainen and
coworkers (1987) also compared liquid-liquid extraction by
magnetic stirring, continuous liquid-liquid extraction for 24
or 48 hours, Ultra-Turrax mixing and Amberlite XAD resin
adsorption. They reported that liquid-liquid extraction by
magnetic stirring for 15 minutes and Ultra Turrax mixing were
not efficient. Continuous liquid-liquid extraction was found
to be comparable to XAD resin adsorption, especially when
extraction times were longer (up to 48 hours). The results
of the present study support the findings of Vartiainen et al.
(1987) since liquid-liquid extraction by magnetic stirring for
24 hours gave results slightly lower than those obtained by
resin adsorption (Fig. 11) . The present results do not
support those of Grabow and coworkers. Their extraction time
was relatively short, and also, they used very large volumes
of water (100 L) compared to organic solvent (520 mL). From
these studies, it appears that at low chlorine or chlorine
dioxide to tryptophan ratios (1:1 or 3:1), Amberlite XAD-2/8
adsorption is superior to liquid-liquid extractions. However,
at high chlorine or chlorine dioxide to tryptophan ratios (7:1
molar ratio), liquid-liquid extractions tend to approach
Amberlite XAD-8/2 adsorption (Fig. 11).
Comparison of aqueous chlorine and chlorine dioxide
A comparison of the mutagenic activities of the two
disinfectants indicated that at the 1:1 molar ratio, chlorine

189
dioxide showed higher mutagenic activity than aqueous chlorine
for both Amberlite XAD adsorption (Fig. 12a) and liquid-liquid
extractions in both strains of bacteria (Fig. 12b). For
comparative purposes, a dose of 100 ¿¿L/plate was used for the
Amberlite XAD-2/8 eluates and 1 mg dry extract/plate for the
liquid-liquid extracts. Statistical analysis of the data at
this ratio showed that the chlorine dioxide reaction products
were significantly (P<0.05) more mutagenic than the aqueous
chlorine reaction products. At the 3:1 ratio, the order of
mutagenic activity was somewhat reversed. The mutagenic
activity was higher in most of the aqueous chlorine reaction
mixtures than those of chlorine dioxide. Statistical analysis
of the data indicated however that at this ratio, the aqueous
chlorine reaction products were significantly more mutagenic
than the chlorine dioxide reaction products in bacteria strain
TA98 and the order was reversed in strain TA100 for the
Amberlite XAD eluates (Fig. 12a). In the liquid-liquid
extracts, the results were not statistically significant (Fig.
12b). Finally at the 7:1 ratio, the reaction products from
aqueous chlorine showed significantly higher mutagenic
activity than chlorine dioxide in both strains of bacteria for
both concentration methods (Fig. 12).
At the low disinfectant to amino acid ratios, (i.e., 1:1
and 3:1), chlorine dioxide reaction products appeared to be
more potent than the aqueous chlorine in most instances.
However, when the concentration of disinfectant was increased

Number of revertants Number of revertants
Figure 12. Comparison of aqueous chlorine and chlorine
dioxide. A) Amberlite XAD 2/8 adsorption (XAD,
dose 100 /iL/plate) , and B) liquid-liquid
extraction (dose 1 mg/plate).

191
further (7:1 molar ratio), the aqueous chlorine reaction
products became more potent than those of chlorine dioxide
(Fig. 12) . The reason for these differences may be explained
by the fact that at the low disinfectant ratios, mainly
oxidation reaction products are produced. The mutagenicities
of the oxidation products have not been studied in any detail.
However, at the highest disinfectant ratio, chlorine dioxide
produces more oxidation byproducts while aqueous chlorine
produces chlorinated reaction products which are more toxic
and/or mutagenic.
Different researchers have compared the mutagenic
activities of aqueous chlorine and chlorine dioxide reaction
products in model systems and in drinking water. In most of
the cases aqueous chlorine produces significantly more
mutagens than chlorine dioxide (Kool et al., 1985), however,
at high chlorine dioxide concentrations (5-15 mg/L),
direct-acting mutagens have been shown to be produced (de
Greef et al., 1980). Kool and colleagues (1985) and de Greef
et al. (1980) showed dose-related increases in mutagenic
activity at increasing chlorine dioxide concentrations in
treated water, especially in S. typhimurium strain TA98.
Other reports in the literature are somewhat conflicting
with regard to the mutagenic activity of chlorine dioxide
reaction products. Rapson et al. (1985) investigated the
mutagenic activity of the reaction products of aqueous
chlorine or chlorine dioxide with tyrosine at increasing

192
disinfectant concentrations. Their results indicated that
mutagenicity peaked at 4 equivalents of chlorine per mole of
tyrosine. The results of the present study are in agreement
with these findings. Also, according to Rapson and colleagues
(1985) replacement of chlorine with chlorine dioxide produced
no mutagens but this study showed otherwise.
From the results of the TLC subfractionation it is clear
that aqueous chlorine reaction products were more potent
mutagens than those of chlorine dioxide. None of the TLC
subfractions of the aqueous chlorine and chlorine dioxide
reaction products appeared similar therefore the mutagenic
activities cannot be compared directly. From their relative
mutagenicity values the least polar of the aqueous chlorine
subfractions (band #5) had a mutagenicity ratio of 35.6 and
19.0 for bacterial strains TA100 and TA98, respectively, while
the corresponding values for the chlorine dioxide sub¬
fractions were 1.5 and 2.6 at the highest doses tested. The
most mutagenic of the TLC subfractions of the chlorine dioxide
reaction products had mutagenicity ratio values of 4.7 and 5.4
for TA98 and TA100, respectively. Other strong mutagens with
very high mutagenicity ratios were detected in the TLC
subfractions of the aqueous chlorine samples while the overall
mutagenicity ratios in the chlorine dioxide subfractions were
relatively low (Tables 22 and 25).

193
Induction of Sister Chromatid Exchange by the TLC
Subtractions of the Reaction Products of Aqueous
Chlorine and Chlorine Dioxide with Tryptophan
Sister chromatid exchange (SCE) has received much
attention in genetic toxicology because of its ability to
detect S-dependent DNA-damaging agents (Latt et al., 1981).
The methodology requires the substitution of thymidine by
5-bromodeoxyuridine (BrdU) or some other halogenated
deoxynucleotide for at least one of two cycles of DNA
synthesis. Chromosomes which replicate in the presence of
BrdU for two cell cycles contain one unifiliary substituted
and one bifiliary substituted chromatid. These differences
can then be used to differentially stain the chromatids. The
molecular basis of its induction has not been fully elucidated
but it is thought to involve the breakage and joining of both
DNA duplexes (one in each sister chromatid) present in a
eukaryotic cell after DNA synthesis. Induction of SCE
indicates a possible mutagenic and/or carcinogenic event.
Like the Ames assay, SCE assays can be performed with or
without the addition of the S-9 mix prepared from the livers
of rats treated with Aroclor 1254. Also both assays are
recommended for prescreening of chemicals for carcinogenic
and/or mutagenic activity.
The results of the SCE assay in the present study
indicated that most of the subfractions of the reaction of
both aqueous chlorine and chlorine dioxide with tryptophan
were capable of increasing the SCE frequencies over those of

194
the control samples (Table 26). There was a linear
dose-related increase in SCE frequencies at all the nontoxic
doses for both aqueous chlorine and chlorine dioxide reaction
products. Except for band B of the chlorine dioxide
subfractions, hiqher doses (1 mg/mL) of the other fractions
were found to be cytotoxic and did not provide enough
metaphases for chromosomal analysis. The S-9 mix was not
included in the assay system because it was previously shown
from the Ames tests that the liver enzyme preparations were
not required for the metabolic activation of the test
compounds. In terms of mutagenic or carcinogenic potency,
the aqueous chlorine reaction products were generally higher
than the chlorine dioxide fractions except for band #4 of the
former fractions (Table 26).
The results of the SCE assays further support those of
the Ames assay. These results were as expected since there
is good concordance between the Ames and other short-term
tests including SCE and chromosomal aberrations (Tennant et
al., 1987). In addition to mutagenic activity in the Ames
test, byproducts of chlorinated drinking water have been
reported to induce both SCE and chromosomal aberrations (CAb)
in vitro in cultured mammalian cells (Liimatainen and Grummt,
1988; Meier and Bull, 1985). The disinfectant chlorine itself
has been reported to induce CAb in mammalian cells (Mickey and
Holden, 1971) while chloramine, administered as the synthetic
disinfectant chloramine-T, induced SCE in vitro in cultured

195
cells (Weitberg, 1987). Chlorinated aquatic humic and fulvic
acids have also been reported to induce SCE and CAb. In a
study by Meier and Bull (1985) , both chlorinated and non-
chlorinated humic acids were capable of inducing SCEs in CHO
cells. Liimatainen and Grummt (1988) also reported that
chlorinated humus-rich surface water induced SCE and CAb in
CHO cells in vitro. However, contrary to the findings of
Meier and Bull (1985), Liimatainen and Grummt (1988) did not
detect any SCE or CAb in the non-chlorinated humus samples.
Other chlorination reaction products such as haloaceto-
nitriles (HAN) have also been reported to induce SCE in vitro
in CHO cells. Bull and Robinson (1985) tested the mutagenic
and/or carcinogenic effects of five commonly found HAN in
water and reported that all produced linear dose-related
increases in SCE frequencies. SCE frequencies increased
linearly with increasing chlorine substitution and replacement
of chlorine with bromine further enhanced SCE induction. The
results of the present study confirm others in the literature.
The results cannot be directly compared since samples, sample
preparation and other parameters were different. However, all
the different results point to the fact that genotoxicants are
produced as a result of water chlorination. The types and
nature of the genotoxicants are varied and diverse and mostly
arise due to the reaction of chlorine with different precursor
substances.

196
Identification of the Reaction Products
From the results of the GC/MS data on the liquid-liquid
extractions, no chlorinated compounds were detected in the
reactions of aqueous chlorine with tryptophan at the 1:1
ratio. The mutagenicity of indolylacetonitrile has been
determined in the Ames' test and found to be nonmutagenic
(Fielding and Horth, 1986). However, it has been shown to be
a precursor of mutagenic activity in cabbage when treated with
nitrite (Wakabayashi et al., 1985). There are no other
reports in the literature concerning the mutagenicity and/or
carcinogenicity of the other indole derivative. The mutagenic
precursor activity of norhaman has been determined in the Ames
test and reported to possess no such activity (Ochiai et al.,
1986). The general mechanism proposed by Dakin (1916) and
other workers (Trehy et al., 1986; Le Cloirec et al., 1985;
van Temelen et al., 1968) may explain the formation of
indolylacetonitrile and some of the products identified in
this study. Tryptophan like some other amino acids readily
react with chlorine to form mono- or dichloramines which
undergo decarboxylation and dehalogenation reactions to form
unstable chloroimine derivatives. The intermediates undergo
further dehalogenation reactions to form nitriles and
depending on the conditions, aldehydes (Fig. 4) . Other
products are possible from the reactions of tryptophan and
chlorine as shown by the general mechanism in Fig. 13.

197
CH7CH—COOH
nh2
Tryptophan
V
CH2C-C00H
NX
H or
Aldehyde
rCH0CN CX2CN
H
Nitrile
Figure 13. Mechanism for the reaction of tryptophan with
aqueous chlorine. X=Halogen. (Modified from Trehy
et al., 1986).

198
When an excess of chlorinating agent was used (7:1
ratio), there were more fragmentation and chlorination
reactions leading to the formation of chlorinated compounds.
The mutagenic activities of some of the identified compounds
such as chloral, 1,1,3-trichloropropanone and 1,1,3,3-tetra-
chloropropanone have been determined. All three compounds
were produced from the reactions of chlorine with humic
substances and found to be mutagenic in S^. typhimurium TA100
without metabolic activation (Fielding and Horth, 1986; Meier
et al., 1986; Coleman et al., 1984; Kringstad et al., 1983).
The mass spectra of 1,1,3-trichloropropanone and 1,1,3,3-
tetrachloropropanone are presented in Fig. 14. It is not
surprising that similar reaction products were formed from
the chlorination of tryptophan since there are some structural
similarities. The chloroketones are known precursors of
chloroform formation and 1,1,3,3-tetrachloropropanone has been
identified as a precursor of MX, a very potent bacterial
mutagen (Padmapriya et al., 1985; Holmbolm et al., 1984).
The mechanism for the formation of the chloroketones is
unclear but it is thought to involve ring chlorination and
subsequent ring opening and further chlorination,
dechlorination and/or oxidation similar to that proposed by
Christman et al., (1983) for alkanoic acids (Fig. 15) or by
Rook (1980) for resorcinol (Fig. 16) . The mechanism of
formation of the quinoline-type compounds is similar to that

Figure 14. Electron impact mass spectra of A) 1,1,3-trichloropropanone and
B) 1,1,3,3-tetrachloropropanone.
199

OH
OH
3HOCI
S^'OH
OH
Cl
CI3C.CO.CCI = CH.CCI2COOH
-C02
f
CI3C.CO.CCI = ch.cci2
HOCI
CI2C.C0.CCI = ch.cci2cooh
OH
HOOC.CCI = CH.COOH 4- CHCI,
OH
HOOC.CCI + CH.CHCI2 + CHCI3
HOCI
HOOC.CCI = CH.CHCI-
Figure 16. Proposed pathway for the formation of chlorinated acetones and
aldehydes from resorcinol (Adapted from Morris, 1978).
200

Figure 15. Proposed scheme for the formation of unsaturated alkanoic acids (Adapted
from Christman et al., 1983).
201

202
of the oxidative decarboxylation reactions reported by van
Temelen et al. (1968) (Fig. 17).
Implications of the Mutagenicity to Carcinogenicity of
the Findings
Because mutagens have been described as possible
carcinogens it is important to determine whether mutagens
identified in drinking water pose a health risk to humans.
Tryptophan and some of its unstable metabolites such as 3-
hydroxykynurenine, have been reported to promote
carcinogenesis and increase bladder tumor yields in rats
pretreated with a nitrofuran (Friedell, 1977). Treatment of
tryptophan with chlorine or chlorine dioxide has been reported
to lead to mutagen formation in model reactions (Owusu-Yaw et
al., 1989; Sen et al., 1989; Horth et al., 1987). It is also
mutagenic upon reaction with nitrite due to the formation of
the nitroso derivative (Ohara et al., 1988) . The pyrrole ring
of tryptophan, like some environmentally significant
heterocyclics, is activated and provides sites for
chlorination and subseguent haloform formation. Studies by
Morris and Baum (1978) indicated that reaction of excess
chlorine with tryptophan produced 18% molar yields of
chloroform at pH 7.5 compared to almost 100% at pH 11. The
prolific chloroform production by tryptophan may explain the
formation of the chloroketones identified in this study. The
chloroketones are known precursors of chloroform formation in

203
CHLCH-C00H
nh2
Tryptophan
CH2CHO
Cl
ch2ch
II
NCI
ch2cho
Quinoline
Figure 13. Proposed mechanism for the formation of quinoline
from tryptophan (Adapted from van Temelen et al.,
1968) .

204
drinking water and since chloroform has already been shown to
be a carcinogen, there is still some concern about its
production in drinking water.
From the safety point of view, it is likely that some of
the identified compounds may contribute to some of the human
cancer incidencies worldwide. However, whether some cancers
arise solely due to water chlorination has not been proven.
Epidemiological data suggest that certain cancers of the
colon, bladder and rectum may be due to water chlorination
(Cantor et al., 1985; Crump, 1983). It is not possible to
substantiate the epidemiological data since they were made
after the fact and other factors such as smoking and dietary
habits were not accounted for. Even though the organic
mutagens are formed in minute quantities their continued
presence in drinking water still poses a health problem over
the long term. Most of the chlorination reaction products
have been tested in bacteria and other mammalian cells but not
in humans or whole animals. Attempts at using whole animal
assays often prove futile due to the fact that most of the
genotoxicants do not require metabolic activation and are
metabolized by the enzymes of the liver into inactive forms
(Meier, 1988). A solution to the problem is to limit human
exposure to these known genotoxicants, find alternative
disinfectants, or sorb the organics from drinking after
chlorination.

CHAPTER VI
SUMMARY
The mutagenicity data presented in this study indicated
that the reaction products from both aqueous chlorine and
chlorine dioxide with tryptophan were mutagenic in the Ames
Salmonella/microsome assay. The compounds were also capable
of inducing SCE in CHO cells. The non-volatile mutagenic
reaction products were mainly direct-acting compounds capable
of causing both frameshift and base-pair substitution
mutations. pH adjustment of the reaction mixture into acidic,
neutral and alkaline fractions indicated that neutral and
acidic compounds accounted for the bulk of the mutagenic
activity. Ethyl ether was found to be more suitable than
either methylene chloride or hexane for extracting the
chemical mutagens from the aqueous reaction mixtures. The use
of Amberlite XAD-2 and XAD-8 resins (1:1 mixture) in one
column was found to be more efficient than the use of either
resin alone and the order of placement of the resins in the
glass column did not have any effect on the amount of mutagens
adsorbed on the column. Also, sample acidification to pH 2.5
prior to resin adsorption was found to increase the recovery
and subsequent mutagenic activity in the acetone eluates.
Amberlite XAD resin adsorption was found to be superior to
205

206
liquid-liquid extraction for the concentration of minute
quantities of organic mutagens from the model reaction
mixtures.
Subsequent TLC separation of the reaction products on
silica gel plates resolved the reaction products into
identifiable blue and green fluorescent bands. Polar
compounds accounted for the bulk of the chemicals present in
the TLC subfractions but the strongest mutagens were
relatively nonpolar. These results were expected since
chemical mutagens are produced in very minute quantities (ppb
levels) in drinking water. Further fractionation of a highly
mutagenic band on another TLC plate produced one fraction
which represented about one-third of total mutagenic activity
in the original fraction.
GC/MS analysis of the reaction products indicated that
no chlorinated compounds were present at the 1:1 ratio even
though some precursors of mutagenic activity were identified.
However, at the 7:1 ratio chlorine to tryptophan ratio, there
was more fragmentation of the tryptophan molecule leading to
the formation of chlorinated byproducts, some of which are
known mutagens or precursors of carcinogenic activity.
The health implications of the mutagenic reaction
products are not clear since drinking water may contain
several chemicals some of which are not produced by
chlorination. The results of the present findings are very
important especially in the food industry where protein and

207
amino acid concentrations are likely to be very high. In
instances where organic matter in food process water is very
high, excess chlorine is used to treat process water for reuse
or eventual discharge into the environment (Robinson et al.,
1981). Such practices are likely to introduce more mutagens
into the environment which may have a serious impact on
aquatic life. No studies have been performed under conditions
of food processing, therefore the full impact is not known.
Also, most of the genotoxicity data on drinking water
chlorination byproducts are done in short-term tests which
makes extrapolation to risk assessment in humans difficult.
The problem may be compounded by the presence of ammonia in
the water which may prevent the destruction of proteins. In
order to alleviate the problem, the use of coagulation and
sedimentation as well as filtration is expected to remove some
but not all of the organic matter in drinking or food
processing water. Finally, the proposed replacement of
chlorine with chlorine dioxide for drinking water disinfection
needs to be examined further since both are capable of causing
mutagen formation in drinking water.

APPENDIX

Figure A-l. Electron impact mass spectrum of oxindole.

Relative Abundance
100
113
33
M/Z 40 80 120 160
Figure A-2. Electron impact mass spectrum of isatin.
o

Relative Abundance
Figure A-3. Electron impact mass spectrum of indolylacetonitrile.
211

Relative Abundance
Figure A-4. Electron impact mass spectra of A) chloral and B) dichloroquinoline.
212

RIC
213
Scan Number
Figure A-5. Reconstructed ion chromatogram of the TLC
subfraction of the reaction products of chlorine
dioxide with tryptophan (band #4). 1) chloral.

Relative Abundance
luu.Ú -i
lito
131.
Figure A-6. Chemical ionization mass spectrum of 1,1,3,3-tetrachloropropanone.
214

Relative Abundance
100.0-
Figure A-7. Chemical ionization mass spectrum of 1,1,3-trichloropropanone.
215

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BIOGRAPHICAL SKETCH
Joe D. Owusu-Yaw, was born on July 15, 1953, in Kumasi,
Ghana. He graduated from Opoku Ware Secondary School, Kumasi,
Ghana, in June 1971 and enrolled at the University of Ghana
in September of the same year where he received a Bachelor of
Science degree with honors in food science and biochemistry
in June 1976. After serving a one-year compulsory National
Service training at the Takoradi Flour Mill, he took a
position as Technical Editor of the Ghana Journal of
Agricultural Science. He enrolled at the University of
Florida in January 1982 and received a Master of Science
degree in food science at the Food Science and Human Nutrition
Department in April 1984. He continued his education at the
same department and is a candidate for the degree of Doctor
of Philosophy to be received in August 1989. He is married
to the former Inza B. Gibson and has a son, Tristian, and a
daughter, Jocelyn.
246

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the deqree of Doctor of Philosophy.
)
r- - •
Cheng-i Wei*
Associate Pi=
Chair
ofessor of Food
Science and Human Nutrition
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the deqree of Doctor of Philosophy.
A
Willis B. Wheeler, Cochair
Professor of Food Science
and Human Nutrition
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
'Jesse F. Gregory,
Professor of Food Science
and Human Nutrition

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
lOCu-—
CS^ai^d'e McGowan
Assistant Professor of Food
Science and Human Nutrition
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of philosophy.
Professor of Entomology and
Nematology
This dissertation was submitted to the Graduate Faculty
of the College of Agriculture and to the Graduate School and
was accepted as partial requirements for the degree of Doctor
of Philosophy.
August 1989
Dean, College of Agriculture
Dean, Graduate School

UNIVERSITY

UNIVERSITY