Analysis of Pesticides in Water. Volume I, Significance, Principles, Techniques, and Chemistry of Pesticides by Afghan, B. K. Chau, Alfred S. Y. Robinson, James W

CRC SERIES IN ANALYSIS FOR
ENVIRONMENTAL CONTROL
Editor-in-Chief
James W. Robinson
Professor
College of Chemistry and Physics
Louisiana State University
Baton Rouge, Louisiana
ANALYSIS OF PESTICIDES IN WATER

Volume I: Significance, Principles, Techniques, and Chemistry of Pesticides
Volume 11: Chlorine- and Phosphorus-Containing Pesticides
Volume 111: Nitrogen-Containing Pesticides
Alfred S. Y. Chau, Senior Editor

B. K. Afghan, CO-Editor
Canada Centre for Inland Waters
Burlington, Ontario
Canada
CHEMICAL ANALYSIS OF
INORGANIC CONSTITUENTS OF WATER
Editor
Jon C. Van Loon
Department of Geology and Chemistry
University of Toronto
Canada
Boca Raton London New York
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Significance, principles, techniques, and chemistry of

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1. Pesticides—Analysis. I. Chau, Alfred S. Y.
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FOREWORD
The assessment of the environmental impact of man's endeavors must be made as

rapidly and painlessly as possible. It is not only aesthetically rewarding to retrieve clean
lakes, rivers, air, and countrysides; it is possible vital to man's continued existence.
Emotional reaction serves no useful long-range purpose, but in fact eventually boom-
erangs and disenchants the public. Nevertheless, it is incumbent upon man t o safeguard
his environment. Clearly, the forces involved are quite beyond our comprehension at
this time. When we are operating from a position of ignorance as we are today, it is
most important to be sure that the risks we take are minimal, although in many cases
a 'fail safe' posture has been adopted which can be extremely expensive. Discussion
on the possible short-term and long-term impacts of various pollutants is valuable but
probably endless. The truth ultimately emerges from reliable data interpreted with wis-
dom and understanding. Analytical chemistry provides the invaluable bridge between
speculation and firm data. We can generate firm data only with reliable analytical
techniques, skilled scientists, and clear minds.
In a n effort t o provide means of collecting and dispersing this information to all
interested parties, we have invited a number of scientists of stature to produce mono-
graphs in their field of expertise. The objective of these monographs is to document
analytical procedures and techniques that are useful to the environmentalists.
In general, three groups of people will be interested in these monographs: industrial
engineers and scientists who are monitoring both liquid and gaseous effluents from
industrial effluents; and environmentalists who are trying to assess pollution levels and
amass data on long-term health effects and other effects of pollution.
This book, collected by Mr. Chau and Dr. Afghan, is devoted t o the broad and
important topic of Pesticides. It examines important facets such as the Significance of
the Problem, the Chemistry of Pesticides, and Principles and Techniques. It will pro-
vide excellent reference material for producers, users, and testing agencies.
J. W. Robinson
Editor-in-Chief
June 1977
EDITOR-IN-CHIEF
J. W. Robinson is Professor of Chemistry and Chairman of the Analytical Division

a t Louisiana State University, Baton Rouge, Louisiana. He earned his degrees at The
University of Birmingham, England (B.Sc., 1949; Ph.D., 1952; D.Sc., 1977) and ob-
tained American citizenship in 1965.
H e worked a t both Exxon Research Company and Ethyl Research Corporation f o r
a number of years before returning to Louisiana State University to join its Chemistry
Faculty.
Dr. Robinson has written more than 130 publications, as well as two texts: Under-
graduate Instrumental Analysis, the 3rd edition of which is currently in press, and
Atomic Absorption Spectroscopy, the 2nd edition of which was published in 1975. H e
is Editor of two international journals, Spectroscopy Letters and of Environmental
Science and Engineering. H e is also assistant editor of Applied Spectroscopy Reviews.
H e is a former chairman of the Cordon Research Conference o n Analytical Chemistry
a n d of the L.S.U. International Symposium on Analytical Chemistry. He is also direc-
tor of the Saul C o r d o n Workship on Atomic Absorption Spectroscopy.
Dr. Robinson is a Guggenheim Fellow and a n Awardee of the Honor Scroll of the
American Institute of Chemists.
PREFACE
Recently, there have been an increasing number of xenobiotic materials entering into
our environment. Many of them are hazardous to human health and to the ecosystem.
Indeed, the problem of environmental protection and pollution control has become
one of modern man's preoccupations.
One prerequisite for decision making in environmental protection and pollution con-
trol is the ability to identify and measure these xenobiotic materials in our ecosystem.
In fact, nearly every phase of environmental protection and pollution control depends
upon analytical data. However, it is not sufficient merely to generate data. These data
must be reliable and truly represent the situation.
Since the analytical data are used for various stages in the activities of international
and national environmental protection and pollution control, the analytical data thus
have far-reaching political, scientific, and financial implications and impact. When
there is no information on the quality of data, the decisions based on them, at best,
are questionable. At worst, if the data are poor, irrational decisions will result. There-
fore, an effective quality assurance program is needed to ensure the reliability of data.
Suitable analytical methodology is the first consideration in an effective quality assur-
ance program for the generation of reliable data.
Unlike the situation for inorganic pollutants, the nature of organic pollutants is
extremely complex and diversified. The number and types of organic pollutants includ-
ing pesticides are also constantly increasing and changing. Due to the numerous vari-
ables (sample matrix, and concentration and types of pollutants), analysis and method
development for these materials is a challenge even to the experienced chemist. In fact,
for the analysis of many organic pollutants in several environmental substrates, suita-
ble methodology is still lacking.
In these three volumes on pesticides, we have tried to present a detailed survey of
the analytical methodology and the essential background information emphasizing the
practical aspects derived from evaluation of literature data and the authors' own ex-
perience. The pros and cons of the different methods, viewpoints, and approaches are
also discussed. Equal amounts of data and discussion from both sides are presented
so that there is sufficient information for readers to derive their own conclusions, even
though they may not agree with us.
The first volume of this series provides background information on pesticides, while
the subsequent two volumes detail analytical methodology on the different classes of
pesticides. These volumes were written for the analyst as well as for university students,
scientists, and researchers for other disciplines. For the latter groups, an attempt was
made, whenever possible, to explain terminology, basic principles, and theories that
might be unfamiliar to them.
We wish to acknowledge the pleasant and fruitful relationships with all the contrib-
utors. The patience, assistance, and understanding of the publisher are greatly appre-
ciated. I wish to thank my wife Linda for typing and retyping the various versions of
my early manuscripts and also for her understanding during the two and one-half years
I spent working on these volumes. A special acknowledgment of gratitude is extended
to M. Chiba, W. Sans, B. Ripley, M. Forbes, and D. McGregor for their valuable
suggestions and critical review of the chapter on the chemical derivatization-gas chro-
matographic techniques.
A. S. Y. Chau, 1981
THE SENIOR EDITOR
Alfred S. Y. Chau is Head, Quality Assurance Methods Section of the Analytical

Methods Division, National Water Research Institute, Canada Centre for Inland
Waters.
He obtained his B.Sc. degree from the University of British Columbia in 1961 and
the M.Sc. degree from Carleton University in 1966.
From 1965 to 1970, Alfred Chau held the position of pesticide analyst in the De-
partment of Agriculture and later joined the Department of the Environment, first as
Head of Organic Laboratories and then as Head, Special Services Section. He has held
his current position since 1980.
Alfred Chau is the General Referee and member of the Association of Official An-
alytical Chemists, a member of the Chemical Institute of Canada, and a task group
chairman of the American Society for Testing and Materials. He is included in Amer-
ican Men and Women of Science and Who's Who in Finance and Industry and is the
recent recipient of the annual Caledon Award for his contribution to analytical chem-
istry.
Alfred Chau has engaged in research in a number of areas. In addition to a manual
of analysis of pesticides in water, and serving as an associate editor for a book on the
analysis of chlorinated hydrocarbons and hydrocarbon, he has published some 90 pa-
pers on the analysis of pesticides and other contaminants in water and in sediment.
Recently, he has been involved in the development of the first Environmental Standard
Reference Materials for organic contaminants such as PCBs in lake sediment.
Furthur, Alfred S. Y. Chau is well known as an accomplished nature artist, being
represented in commercial galleries across Canada. His works are in many private and
permanent collections including the Dofasco Canadian Art Collection and the Beckett
Collection.
THE CO-EDITOR
B. K. Afghan is research scientist in environmental analytical chemistry in the Ana-

lytical Chemistry Research Section, Canada Centre for Inland Waters, Burlington,
Ontario.
Dr. Afghan received the B.Sc. degree from Sind University, Pakistan, in 1962, and
the D.I.C. and Ph.D. degrees in analytical chemistry from the University of London
in 1964 and 1969, respectively.
Following positions in research at Dalhousje University (1966 to 1968) and the Uni-
versity of Montreal (1968 to 1969), Dr. Afghan served as a research scientist in analyt-
ical methods development in the Department of Energy, Mines, and Resources in Ot-
tawa and has been with the Canadian Centre for Inland Waters since 1972.
Dr. Afghan is a fellow in the Chemical Institute of Canada, member of the editorial
board of the Canadian Journal o f Spectroscopy, and chairman of the task group of
the American Society of Testing and Materials. His research has been concerned with
modern polarographic and electroanalytical techniques, high speed liquid chromatog-
raphy, atomic and molecular absorption and fluorescence spectroscopy, trace analysis,
and environmental analytical chemistry.
Dr. Afghan has published more than 50 research papers over his research career in
the areas of automation, atomic/molecular spectroscopy, luminescence, nutrients,
heavy metals, trace organics, anipestieide residues.
CONTRIBUTORS
Professor John W. ApSimon, P h D . Derek Muir, Ph.D.

Department of Chemistry Research Scientist
Carleton University Freshwater Institute
Ottawa Department of Fisheries and Oceans
Canada Winnipeg, Manitoba
Canada
Walter A. Glooschenko, Ph.D.
Research Scientist Brian D. Ripley
Aquatic Ecology Division Chemist, Carbamates and Fungicides
National Water Research Institute Provincial Pesticide Residue Testing
Burlington, Ontario Laboratory
Canada Ontario Ministry of Agriculture and
Food
Raj Grover, Ph.D. c/o University of Guelph
Section Head Guelph, Ontario
Herbicide Behavior in the Environment Canada
Research Station
Agriculture Canada George J. Sirons
Regina, Saskatchewan Chemist, Herbicides
Canada Provincial Pesticide Residue Testing
Laboratory
Fred K. Kawahara, Ph.D. Guelph, Ontario
Chemist Canada
Environmental Monitoring and Support
Laboratory Allan E. Smith, Ph.D.
Office of Research and Development Research Scientist
Environmental Protection Agency Agriculture Canada
Cincinnati, Ohio Research Station
Regina, Saskatchewan
Hing-Biu Lee, Ph.D. Canada
Chemist
Analytical Methods Division
National Water Research Institute W. M. J. Strachan, Ph.D.
Burlington, Ontario National Water Research Institute
Canada Canada Centre for Inland Waters
Burlington, Ontario
R. James Maguire, Ph.D. Canada
Research Scientist
National Water Research Institute Kazuyuki Yamasaki, Ph.D.
Environmental Contaminant Division Research Fellow
Department of Environment Department of Chemistry
Burlington, Ontario University of California at Los Angeles
Canada Los Angeles, California
ANALYSIS OF PESTICIDES IN WATER
Volume I
SIGNIFICANCE, PRINCIPLES, TECHNIQUES, AND CHEMISTRY O F
PESTICIDES
Environmental Impact and Significance of Pesticides

Basic Principles and Practices in the Analysis of Pesticides
Positive Identification of Pesticide Residues by Chemical ~erivatizationTechniques
The Chemistry of Cyclodiene Insecticides
Volume I1
CHLORINE- AND PHOSPHORUS-CONTAINING PESTICIDES
Organochlorine Pesticides
Organophosphorus Pesticides
Phenoxy Alkyl Acid Herbicides (CPH)
Volume I11
NITROGEN-CONTAINING PESTICIDES
Carbamates
The Substitute Urea Herbicides
Triazine Herbicides
TABLE OF CONTENTS
Volume I
Chapter l
Environmental Impact and Significance of Pesticides. . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
W. M. J . Strachan, W. H. Glooschenko, and R. J. Maguire
Chapter 2
Basic Principles and Practices in the Analysis of Pesticides. . . . . . . . . . . . . . . . . . . . . . .25
A. S. Y. Chau and H. B. Lee
Chapter 3
Positive Identification of Pesticide Residues by Chemical Derivatization
Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
A. S. Y. Chau
Chapter 4
The Chemistry of Cyclodiene Insecticides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
J. W. ApSimon and K. Yamasaki
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Volume I 1
Chapter 1
ENVIRONMENTAL IMPACT AND SIGNIFICANCE OF PESTICIDES
W.M. J . Strachan. W.A. Glooschenko. and R .J . Maguire
TABLE OF CONTENTS
I. Introduction ........................................................ 2
I1. Pesticide Types and Properties ......................................... 2

A. Organochlorines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
B. Organophosphates ............................................. 3
C. Carbamates ................................................... 4
D. Phenoxyalkanoic Acid Derivatives ............................... 4
E. SubstitutedUreas .............................................. 5
F. Triazines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
111. Transport and Movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

A. Atmosphere .................................................. 6
B. Water . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
C. Sediments .................................................... 9
IV . Accumulation ...................................................... 11
V. Degradation ....................................................... 12
V1. Modeling .......................................................... 15
V11. Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
VIII . Monitoring ........................................................ 17
References ............................................................... 18
2 Analysis o f Pesticides in Water
I. INTRODUCTION
The use of the term pesticides in the title of this volume would seem to encompass
a very wide range of materials. The broadest dictionary' definition of pest, if applied
here, would indicate that all organic compounds used against troublesome persons,
animals, or things are to be considered. In fact, an examination of the other chapter
headings in this volume shows that the concern is with insects and weeds, as indicated
by the inclusion of the organochlorine, organophosphorous and carbamate pesticides
and the phenoxyalkanoic acid, urea, and triazine herbicides. The normal usage of such
materials involves their direct application to the environment and this, coupled with
their biocidal nature, requires that considerable effort be expended to understand their
impact, movement, and transformations in the ecosystem. Since the topic of this vol-
ume is water pollution, it is to this part of the biosphere that attention is focused in
this chapter.
Pesticide reactions of aquatic environmental interest can be divided into two broad
types - abiotic and biological. In the former, hydrolysis and photochemical processes
are of greatest general concern; in the latter, the possibilities are extensive. Many of
the processes of both types result in the same products and, while the rates of the
abiotic processes can be determined in the laboratory and reasonably applied to the
environment, those of a biological nature are dependent on the nature, numbers, and
adaptation of the organisms involved and do not readily lend themselves to meaningful
laboratory determination. Some qualitative definition of the nature of microorganisms
in appropriate environmental compartments is possible, e.g., sulfur-reducing bacteria,
nitrogen-fixing bacteria, etc., but this will be largely inadequate for the prediction of
environmental rates without a knowledge of the behavior of some reference sub-
stance(~)in both the environment of concern and in the test system. Even then, accli-
mation to the pesticide of concern will be an important f a ~ t o r . ~For
. ~larger
. ~ organisms
such as plankton, invertebrates, and fish, magnification of persistent materials within
a food chain is, for some of the substances in this chapter, a major focus for environ-
mental research. It is particularly in the lipid that the parent substances
or their persistent metabolites are likely to occur either as themselves or as a conjugated
moiety incorporated into some biological m a t r i ~ . ~ . ~Therefore,
."' any monitoring activ-
ity should take account of and report on this aspect as well as the actual level of the
substances in the organisms. These various considerations all require that appropriate
analytical procedures be available, and such procedures are the foci of the other chap-
ters in this volume. In this chapter, however, it is intended to give an idea of the
environmental concerns of the six classes of pesticides mentioned.
11. PESTICIDE TYPES AND PROPERTIES
A. Organochlorines
Members of this class of pesticides have given rise to the greatest environmental
concern because of their great stability in the aquatic ecosystem. There are several
common structural types among this group; some of these, along with prominent ex-
amples, are
Volume I 3
hexachlorocyclo- hexachloro- diphenyltrichloro- chlorinated cyclopenta-

hexanes (a,p, y benzene ethanes (DDT, diene adducts (aldrin,
isomers of BHC) methoxychlor) dieldrin, mirex, endosul-
fan, heptachlor, endrin)
In general, they are resistant to hydrolysis, and those that undergo photochemical re-
action (usually with photosensitizing) tend to form compounds with persistence com-
parable to, or greater than, their parent corn pound^.^^.^^.^^ They are also insoluble in
water and the environmental concerns have focused mostly on their potential for bio-
logical accumulation.
Biologically, reductive dechlorination and dehydrochlorination in the DDT-type pes-
ticides and the BHC isomers are the major metabolic routes for those compounds,
resulting in highly stable p r o d u ~ t s . ' ~
For
~ ' the
~ cyclopentadiene adducts, dechlorination
of vinylic chloride atoms and e p o ~ i d a t i o n ' ~ ~are " ~ the
' ~ ~most common processes,
depending upon the oxidizing ability of the medium and organism. Epoxidation is
particularly predominant for aldrin (+dieldrin) and heptachlor (+heptachlor epoxide).
B. Organophosphates
These compounds are generally a concern in more localized situations than are the
organochlorines. They are generally more acutely toxic than their chlorinated alterna-
tives, but their persistence is much less. They are also much more soluble in water and
as a consequence are less likely to accumulate in biological tissue. Three major types
of this class of pesticides and common examples are
S 0 S
U 1
I 1
I
(RO)2 -P-OR' (RO), -P-SR' (RO), -P-SR'
phosphorothionates phosphorothiolates phosphorodithioates

(parathions, fenitro- (demeton) (malathion, azinphos-
thion, diazinon) methyl)
By far the most common is the phosphorothionate pesticide. Completely oxygenated

analogs of the above, the phosphates, are known but are much less stable environmen-
tally although they are even more toxic.21
With all the above pesticide types, there are three organic ester groups, two of which
are usually methyl or ethyl. The selection of the third group usually governs the partic-
ular persistence and toxicity, with functional groups R' increasing toxicity and persist-
ence by hindering (sterically and electronically) hydrolysis at the P atom. The third
ester group is frequently an acid (phenolic or an active hydroxyl) function and is sub-
ject to alkaline hydrolysis. However, under environmental conditions, the purely
abiotic hydrolysis is very slow and often insignificant when compared with biological
degradation.
Photochemically, a common occurrence is the conversion of the P S bond to a P
+
+ 0.12 This results in a hydrolytically much less stable entity which is of lesser environ-
mental concern as a consequence. The same process, however, also occurs in the bodies
of aquatic organisms with mixed-function o x i d a s e ~ ,where~ ~ , ~the
~ greater toxicity of
4 Analysis of Pesticides in Water
the oxygen analogs may affect the organism. Photochemically, it is also possible to
oxidize the side chains attached to aromatic ester groups or other easily oxidized groups
in the ester f u n c t i ~ n ; *P~-. ~0~- aryl cleavage also occurs.27
Biologically, the "organophosphorous" pesticides act by interfering with enzyme
systems, mainly acelylcholinesterase.28~29 The oxygen analogs have little stability them-
selves so that the concern is largely for the parent pesticide acting through the oxygen
analog.25Biological reaction generally results in release of the most acidic ester group,
although reduction of the nitro functional group in an aromatic phenolic ester has
been o b s e r ~ e d . ~ ~ . ~ ' . ~ ~
C. Carbamates
These compounds are all derivatives of carbamic acid with the general formula
When R is an aromatic phenolic ester group and R' and R" are H or alkyl groups, the
compounds are used as insecticides. When an aromatic ring is a substituent on the
nitrogen atom, the compounds are generally herbicides; sulfur analogs of the insecti-
cide thiocarbamates are used as fungicides.
These compounds, particularly the insecticides with a monoalkyl substituent on ni-
trogen, are relatively easily hydrolyzed and in general are not considered an environ-
mental problem, due t o their low persistence.
D. Phenoxyalkanoic Acid Derivatives

Compounds of this type are used as herbicides, both on agricultural land and in
controlling weeds in nonproductive areas such as roadsides and electrical transmission
lines. Their general chemical structure is
where X is 1-3 (usually in the 2, 4, and 5 ring positions), y is 1-3, and R may be any of
a number of structures ranging from metal and ammonium ions (both substituted and
unsubstituted) to a wide variety of organic alcohols. The nature of the R group will
be the major factor in determining the behavior of these compounds. The "salts" are
water soluble and move with that phase; the organic esters are not and behave very
differently. The latter will undergo hydrolysis to yield the parent acid, usually with
half-lives in the order of a few h o ~ r s .Photochemically,
~ ~ . ~ ~ these compounds all form
the substituted phenols with varying ease as well as undergoing substitution reactions
of hydroxyl for the ring chlorines.34
Biologically, the phenoxyalkanoic acid compounds are not particularly stable. In
the major target area (soils), they are fairly readily broken down;3s in the aquatic en-
vironment, the anaerobic microorganisms which often predominate are not always ef-
fective in degrading these c o r n p o ~ n d s In
. ~aerobic
~ ~ ~ ~ situations, phenoxyalkanoic acid
derivatives d o not seem to persist.
One of the environmental concerns from the use of 2,4,5-trichlorophenol derivatives
is the potential presence of TCDDs (particularly 2,3,7,8-tetrachlorodibenzo-pdioxin),
which may be present as by-products of the preparation of the p h e n ~ l . This ~ ~ .sub-
~~
stance is teratogenic and mutagenic; its chronic effects4' are well known; and it seems
to be per~istent.~' While it only occurs in trace quantities, its presence has recently
been noted in the environment and its occurrence should be watched for and is cause
for concern.
Volume I 5
E. Substituted Ureas
The substituted ureas form another common class of biocides. They are primarily
herbicides and have the general chemical structure
0
U
RNH-C -NR'R1'
where R is most frequently a substituted (chlorinated) aromatic ring and R', R" are
hydrogen, alkyl, or alkoxy groups. Their water solubility is generally low, but as a
result of their facile environmental reactions, they do not present problems of persist-
ence and accumulation.
Environmentally, these compounds are moderately unstable, undergoing dealkyla-
tion and dearylation fairly r e a d i l ~ and
~ ~ hence,
, ~ ~ environmentally, they are of local
concern only in the area and period of application.
F. Triazines
The basic chemical structure for this type of herbicide is
In general, the two amino functions are monosubstituted with alkyl groups, but this
is not always the case. Similarly, substitution at the X position of the ring with C1 is
normal, although herbicides with alkoxy and alkylthio groups are known. The most
prominent member of this group is atrazine (X = Cl, R = i-propyl, ethyl), which is
used in great quantities as a pre-emergent herbicide in corn fields.
The hydrolysis of the 2-chloro substituent, t o give the Zhydroxy triazine analog
takes place readily in conditions relevant to the e n ~ i r o n m e n t .Photochemical
~~,~~ reac-
tions also give the same products, but the rates are not significant under environmental
condition~.~~
Biologically, dealkylation and substitution are the major routes of metabolic break-
doWn.47.48.49 In soils, dealkylation appears to be the major route with considerable
persistence of the resulting products. As these products retain the active ring structure,
it is apparent that surveillance programs for the parent compound should include the
metabolites as well. In aerobic aqueous systems, including moist soils, substitution of
a hydroxyl group for the chlorine atom precedes ring cleavage and complete degrada-
t i ~ n . ~In' situations where this is unlikely, such as in anaerobic sediments, the break-
down is likely to be slow: hence triazines in these environments may be of concern.
111. TRANSPORT AND MOVEMENT
Pesticides enter the water from various sources. E d w a r d ~reports

~ ~ . ~ major
~ sources
to include (1) runoff from agricultural lands, (2) direct entry from spray operations,
(3) industrial effluents, (4) sewage effluents, ( 5 ) spraying of cattle, and (6) dust and
rainfall. In water, the residues and their degradation or transformation products are
distributed between the truly dissolved form and those incorporated into sediments,
benthic invertebrates, aquatic plants, plankton, aquatic invertebrates, suspended det-
ritus, and fish. Pesticides can leave aquatic systems by volatilization or CO-distillation,
as residues in fish which are eaten by man, birds and animals, or by degradation,
Analysis o f Pesticides in Water
FIGURE 1. Dynamic movement of pesticides in the aquatic en-

vironment.
burial in sediments, or outflow. Figure 1 shows the dynamic state of affairs in aquatic
ecosystems which is only a part of the more comprehensive scheme describing the
movement of a pesticide between environmental compartment^.^' All of the indicated
materials may be mobilized, and it is their transport which determines the resulting
distribution of the residues. In a practical sense, their presence in water, sediment, and
the atmosphere will be the major factors determining their availability, and these are
addressed here.
A. Atmosphere
Pesticides have been detected in aquatic ecosystems remote from recognizable
sources. Transport by ocean currents cannot explain such distribution, and aerial
transport and fallout of such compounds has been i m p l i ~ a t e d . The ~ ~ . ~reviews
~ of
Bergesteinsson and Baier,54 W h e a t l e ~ ,and ~ ~ Makhon'Kos6 discuss these sources in
more detail. Other papers dealing with more specific aspects of aerial input include
Hage,57S p e n ~ e rand
, ~ ~L l o y d - J a m e ~ . ~ ~
Pesticides may be transported for long distances. Risebrough et found trans-
Atlantic movement of DDT via the tradewinds, while Cohen and Pinkerton traced
transport of such pesticides as DDT, DDE, chlordane, RonnelB, heptachlor epoxide,
2,4,5-T, and dieldrin from west Texas to Cincinnati via a dust storm.61Transcontinen-
tal DDT movement has also been indicated from a spray program in the U.S. Pacific
northwest area to have been deposited in New York.62
Definite geographical differences occur in atmospheric fallout. Stanley et al.63sam-
pled air from nine U.S. cities and analyzed primarily the particulate fraction for 19
pesticides and their metabolites. Levels ranged from 0.1 to 2520 ng/m3 with only DDT
found at all locations. Organophosphates occurred mainly in the southern U.S. with
highest levels in Florida; lowest levels occurred in urban areas. Maximum levels were
found in summer and were attributed to agricultural usage. In the Great Lakes re-
g i ~ n rain has been analyzed and found to contain the pesticides lindane, a-BHC,
, ~ ~
DDT, and degradation products a- and p-endosulfan, dieldrin, and methoxychlor, all
at mean concentrations less than 0.03 pg/l. Snow contained only a-BHC, DDT, and
methoxychlor at 0.001 to 0.002 pg/l.
Pesticide deposition from the atmosphere is worldwide. A study of DDT inputs in
Volume I 7
a southern Swedish lake was made by S ~ d e r g r e nwho

, ~ ~ found atmospheric input to
be the major source. From August to October, 3600 ng/m2 of DDT and its degradation
products were trapped in fallout screens with particulate matter being a major deposi-
tional mode. In addition to the above examples, there are many reported instances of
deposition in widely separated areas such as Hawaii,66 Japan,67C a l i f ~ r n i a , ~and
' the
British Isles.69
B. Water
Analysis of natural waters for pesticides has been done in many studies. Before one
can interpret results of such analyses, it is important to understand the physical-chem-
ical behavior of the pesticides in water. Their solubility in water can be a function of
various parameters besides chemical structure of the compounds - pH, temperature,
salt concentration, and organic matter concentrations in the water in particular are
important. Solubility can also vary extensively within a given class of pesticides. For
example, Edwards70gives the following ranges of solubilities:
1. Organochlorines - 0.0012 mg/l for DDT to 7 mg/l for lindane

2. Organophosphates - 24 mg/l for parathion to 2500 mg/l for dimethoate
3. Carbarnates - 40 mg/l for carbaryl to 6000 mg/P for aldicarb
4. Herbicides - 5 mg/l for simazine to 890 mg/l for 2,4-D
It is apparent that a pesticide may be present in water in a dissolved form or as a

precepitate if solubility criteria are exceeded. Pesticides may also be associated with
organic surface films. Seba and Corcoran7' studied such a phenomenon and found
that such slicks contained pesticides such as DDT, dieldrin, and aldrin in the low mi-
crogram per liter range while seawater below the surface had less than 1 ng/l. Wershaw
et al.72 also noted that natural organic compounds such as humic substances could
solubilize pesticides such as DDT or 2,4,5-T.
Particulates suspended in the water column may also contain pesticide residues.
Odum et found that DDT and its metabolites accumulate in organic plant detritus
mainly in the 250- to 1000-pm diameter size fraction at levels 20 to 50 times higher
than in living plants. These particulates are available for ingestion by biota such as
crabs and by fish feeding on the suspended materials. Pierce et studied p, p'-DDT
adsorption to suspended particulate matter in seawater. Humic particulates had a
greater adsorptive capacity than that of clays; lipid materials showed the highest ad-
sorption. Plankton in water also concentrate pesticide^^^ and must be removed by fil-
tration from water if only soluble pesticides are to be determined. These particles and
the water in which they are suspended move. Their deposition and resuspension be-
comes important, and information on likely areas for such events is necessary in inter-
preting the results of analytical surveys.
In general, waters contain pesticide concentrations reflecting regional and local
usage. Lichtenberg et al.76sampled 529 U.S. waters and reported mainly DDT and its
derivatives, and dieldrin, with levels often exceeding 0.05 pg/l. The highest frequency
of occurrence was found in the southern and eastern U.S., reflecting more intensive
agriculture and higher population densities. Zabik et studied the level of pesticides
in the Red Cedar River in Michigan. In general, pesticide concentrations were highest
in June, while those of their degradation products were highest in April, indicating
winter degradation and subsequent flushing into the river during thaw. Levels were
greater in river sediments than in water. In this instance, the authors found that the
major source was from a wastewater treatment plant, not agriculture.
Truhlar and Reed78 studied pesticide residues in four Pennsylvania watersheds re-
flecting forest, general farming, residential areas, and orchards. DDT and its degra-
dation products were the most common pesticides found in soils except in the forest
watershed where none was detected. Pesticides found in the water included dieldrin,
chlordane, heptachlor epoxide, lindane, and aldrin in order of decreasing concentra-
tion. The herbicides 2,4-D, SilvexB, and 2,4,5-T were also present at times. Median
DDT levels were highest in the orchard stream at 140 pg P-' followed by 22 pg P-' in
the residential stream, 0.6 pg 1-' in the forested stream, and below detectability for
the general farm area. However, even though the median level was higher in the or-
chard stream compared to the residential stream, much higher peak values were re-
ported in the latter during storm runoff.
Western U.S. waters have also been studied in terms of pesticide concentrations. In
a study of selected western U.S. streams, Schulze et found DDT to be the most
common pesticide and 2,4,5-T the most common herbicide, but levels of both were
less than 1 pg/P. The most frequent pesticide occurrences were found in the Gila River,
which drains areas of intensive agriculture in Arizona. In these streams, organophos-
phates such as parathion, methyl parathion, and diazinon were also detected. The au-
thors also found that the highest pesticide levels occurred in streams containing the
highest suspended sediment levels.
In the midwestern U.S., a study was done on Iowa waters for atrazine, DDT, and
dieldrin.80 The herbicide atrazine had the highest concentration levels, reflecting both
its large use in corn crops and its high water s o l ~ b i l i t yHigh
. ~ ~ levels of these com-
pounds were found after rainfall events. These authors also caution against compari-
son of levels in streams due to variables such as sampling time, climatic history, soil
type, sediment load, and time of pesticide application. Another related water sampling
problem is degradation. For example, Ginn and Fishers studied pesticides in waters
from rice fields where aldrin had been applied. This compound degraded quickly to
dieldrin, a process favored by environmental conditions such as high temperature, high
oxygen saturation of water, low pH, high Eh, and soil structure.
Gummers2 carried out a similar survey in western Canadian waters in which the
highest levels reported were for the compounds 2,4-D, 2,4,5-T, lindane, and a-BHC.
There were limited reports for dichloroprop, aldrin, and p-endosulfan. Atmospheric
precipitation was a major reported source for the lindane and BHC, and the highest
herbicide levels were found in agricultural areas which exhibited a seasonal pattern
related t o use. Large urban centers such as Winnipeg, Regina, and Edmonton were
also major sources.
The Great Lakes have also been studied in terms of pesticides in water as reviewed
by Harris and Miles.83 In general, large cities such as Chicago appear to be major
sources of compoundss4 with sewage effluents being high in such compounds as DDT
and methoxychlor. Agricultural usage is also a source,s5 with compounds such as DDT,
dieldrin, leptophos, chlordane, endrin, and heptachlor epoxide being detected in
waters draining such areas. Open waters of the lakes only indicated trace, nonquanti-
fiable concentration^.^^
In terms of marine waters, little has been published, except the work of Mac-
Gregor," who reported sewage treatment plants in southern California to be a major
source of DDT and its degradation products to nearby coastal waters.
Recent studies indicate some pesticides may currently have reduced concentrations
in waters due to bans on their use. Mattrawas did a study in southern Florida and
found the frequency of detectable organochlorine residues in surface waters to be de-
creased from 1961 to 1972. For example, dieldrin dropped from 22 to 15, DDT from
81 to 1.2, DDE from 23 to 3.1, and DDD from 15 to 3.8 pg/l. The major source of
these pesticides was from the Everglades agricultural area, and transportation was by
sediment movement due to currents.
Studies have also been made related to pesticides in drinking water. Schafer et al.89
Volume I 9
analyzed 500 raw and finished water samples from the Mississippi and Missouri River.
The pesticide most frequently detected was dieldrin (40% of samples), followed by
endrin, p, p'-DDT and p, p'DDE (30%) and chlordane (20%). Rarely found were
aldrin and heptachlor, while toxaphene and methoxychlor were not found at all. Water
treatment procedures have varied efficiencies of removal of pesticides, but can often
lead to production of products of increased t o x i ~ i t y . ~One
~ . ' ~source of such materials
may be the pesticide manufacturing plants themselves. The finding of mirex in Lake
Ontario fish far from usage of that pesticide is a good example of this, as the plant
producing this substance was located at Niagara Falls, New York.91Transport of this
pesticide/flame retardant is associated with sediment m ~ v e m e n t . ~ '
Pesticides have also been reported for waters of the Rhine River in Europe where
serious fish kills due to endosulfan have occurred.93 Other studies on this river indi-
cated that such organochlorines as y-BHC, lindane, and HCB were nearly always pres-
ent, while heptachlor, heptachlor epoxide, aldrin, dieldrin, endrin, and DDT were oc-
casionally found, usually at levels lower than 0.1 pg I-'; endosulfan averaged 0.10
pg/L.94 The Rhine also contained less persistent pesticides such as malathion,
parathion, dimethoate, diazinon, and carbaryl at levels of 0.01- to 0.04 pg I-' with
carbaryl being the highest. Other areas reporting pesticides in water include Africa9=
and the Caribbean.96
C . Sediments
The sediment component of aquatic ecosystems can be an ultimate sink of pesticides;
suspended particulates entering slower moving waters such as larger water bodies settle
out, and their associated pesticides are added to the existing sediment component.
Biota, through death and subsequent decay, can be incorporated into sediment organic
fractions and their adsorbed pesticides again added to the content of sediments. Direct
partitioning of pesticides between water and sediments may also occur. All such pesti-
cides may be biochemically transformed by the large biomass of microbes associated
with the sediment/water interface, or may remain available to biota such as inverte-
brates or fish which feed on the benthos of the sediments. The question of bioavaila-
bility of pesticides bound to soils or sediments is of major concern, especially whether
or not a normally nonpersistent organophosphorus pesticide such as parathion may
become bound to sediments and slowly released to organisms.
Relatively little research has been performed on factors influencing the incorpora-
tion of pesticides by sediments. Related information on soil/pesticide interactions may
be pertinent, but it should be noted that sediments tend to be poorly oxygenated when
compared with soils, and hence results from one are not directly applicable to the
other. Several factors have been found to influence adsorption of pesticides to soil.
These include (1) the nature of the pesticide, including molecular structure, charge
characteristics, and water solubility; (2) nature of the soil colloids such as clay content
and type, presence of amorphous inorganic matter, pH and cation content, and or-
ganic matter content; and (3) age (degradation state) of residues. Other factors such
as soil temperature and moisture are certainly important. Research carried out on sed-
iment/pesticide interactions illustrates this. Lotse et al.98 studied the adsorption of
lindane by lake sediments. The most important factors affecting that process were
found to be (1) concentration of suspended sediments, (2) clay content, (3) lindane/
sediment ratio, (4) organic matter content, and (5) lindane concentration. Another
studyP9concerned with sorption and desorption of dieldrin, heptachlor, and DDT by
sediments found that pH was important, while temperature, salt content, and soluble
organic matter content were less so. Boucher and Leeloostudied uptake of lindane and
dieldrin by sands and found no pH or temperature effects; however, natural organic
matter decreased uptake of dieldrin, but not lindane. Wang et a1.I0' studied adsorption
of parathion by lake sediments and found that removal of naturally occurring organic
compounds increased parathion uptake, possibly by exposing new adsorption sites.
The role of bacteria associated with sediments also cannot be neglected. Leshniowsky
et a1.'02 found bacterial flocs associated with sediments which had concentrated diel-
drin.
Sediments can, therefore, be considered as a complex matrix of inorganic compo-
nents (such as clays, sands, and other minerals), particulate and dissolved organic com-
pounds, associated biota, particularly bacteria, and dissolved interstitial cations and
anions. All of these factors make it hard to generalize results of particular experiments,
especially as sediments may also vary in their state of oxygenation, both on a temporal
and spatial basis.
A more practical aspect of sediments is their use in monitoring the influence of local
land use upon water bodies. Agricultural usage alone cannot be invoked as the source
of pesticides to sediments. For example, Wiersma et al.lo3 studied pesticides in soils
from eight U.S. cities. DDT and its degradation products were predominantly found;
city soils were found to be higher in such residues than agricultural areas, especially
lawn areas compared to unkept areas. Another survey by Carey et al.lo4 of 14 U.S.
cities found the same general results, with organochlorine pesticides in city soils greater
than crop lands.
Several studies on pesticides in sediments of the Great Lakes basin are illustrative
of the movement of these compounds. Miles and H a r r i ~ ' studied
~ ~ ~ ' DDT
~ ~ and dieldrin
distribution in Big Creek, a stream entering Lake Erie. Very little DDT was found in
the waters of Big Creek, no more than 0.067 ~ ( g / l however,
; sediments contained up
to 0.44 pg/g, and fish 1 >>g/g. The creek was found to discharge an average of 50 g/
week of DDT to Lake Erie. Ratios of TDE/DDE were greater than 1 in sediments,
indicating degradation, and the ratio increased linearly downstream from the source,
such behavior being consistent with the water solubility of the two compounds. Other
pesticides detected in these sediments were dieldrin and diazinon.
In terms of sediment transport of pesticides, river flow is very important. Miles et
al.85found that most DDT and dieldrin transported by Big Creek occurred during peak
spring runoff. Land use also affects sediment concentrations of pesticides. Frank et
al.''' studied four areas of southern Ontario with differing land use patterns, one ag-
ricultural, two mixed agriculture and recreation, and one recreational area on the Ca-
nadian Shield. Of particular interest was the finding that sediments contained approx-
imately equal concentrations of DDT and its degradation products, except for the
recreation area where DDT was slightly higher. The same was found for fish caught
in the four areas, indicating the possibility of indiscriminate pesticide applications by
cottage owners for mosquito and black fly control or general atmospheric deposition.
Another instance in which sediment transport was implicated was a study on the
organochlorine distribution in sediments of Rattray Marsh, a small wetland located
on the shore of Lake Ontario near Toronto, Ontario.'08 Sediments contained con-
firmed levels of the pesticides and its degradation products DDE and TDE, plus chlor-
dane isomers. Sheridan Creek, the major tributary entering the marsh, appeared to be
the major source of such inputs to marsh sediments, but atmospheric input was not
completely ruled out. The assessment of inputs was based on the fact that highest levels
of these compounds were found in the areas of the marsh where most rapid sedimen-
tation o f Sheridan Creek suspended sediments was occurring. An analysis of suspended
material from the creek confirmed their presence there.
In the Great Lakes, Glooschenko et investigated sediment pesticides including
16 organochlorine compounds and 17 organophosphorus compounds in Lakes Huron
and Superior and Georgian Bay. Only DDT, DDE, TDE, and dieldrin were found,
with highest concentrations occurring in deeper basins where fine sediments were ac-
Volume I 11
cumulating. Frank et al.'09 studied pesticides in Lake Ontario sediments, and p, p'-
DDT and its degradation products were the major compounds found with an average
concentration of 42 pg/g; some residues of heptachlor expoxide, chlordane, and en-
dosulfan were also noted. Again, the highest concentrations were found in depositional
basins. A noteworthy instance of sediment associated pesticide movement is the pattern
of mirex in Lake Ontario. Holdrinet et al.92found, in an analytical survey of surface
sediments, that the deposition of this pesticide/flame retardant conformed well to
south shore currents in the lake with indicated inputs being the Niagara and Oswego
rivers, on both of which were found to be sited likely industrial sources.
In Lakes Erie and St. Clair, the major pesticides again were DDT and its degradation
products with heptachlor epoxide and endosulfan being found less frequently."O Spa-
tial distribution patterns were determined which identified possible sources such as the
Detroit River and local agricultural areas adjacent to the lake. Loadings were calcu-
lated to be 1.0 metric ton year-' for DDE and TDE, and 45.0 kg year-' for heptachlor
epoxide. The most prominent parameter affecting pesticide concentration was sedi-
ment organic carbon content with clays giving a poor correlation.
Studies on pesticides in marine sediments are limited to coastal areas. For example,
Hom et al."' studied DDE in a dated sediment core from the Santa Barbara Basin off
the California coast. The compound was first noted around 1952 and observations
increased up to 1967 when a deposition rate of 1.9 X 10-4g m-2 year-' was calculated.
The major source appeared to be Los Angeles County ocean sewage outfall. Law and
goer lit^"^ studied pesticides in sediments of 26 streams tributary to San Francisco
Bay, California. Chlordane was the most common pesticide, occurring in 92% of sam-
ples; its degradation product, nonachlor, was also detected. DDT compounds were
detected in only 8% of samples. In the Vancouver, British Columbia, area, DDT com-
pounds and chlordane were the major pesticides found in sediments,l13 and in the lower
St. Lawrence River sediments contained mainly DDT compounds with a- and y-BHC,
heptachlor, and dieldrin, all at less than 0.035 ~ g / g . " ~
Organochlorines have also been detected in sediments far removed from known
usage. Salt marsh sediments in subarctic James Bay, Canada, were found to contain
trace amounts of DDT, DDE, endrin, lindane, and methoxychlor with atmospheric
input as the major source.53While data on levels of pesticides in sediments is needed
in evaluating their fate and likely persistence in the aquatic environment, an area which
is seldom studied but very necessary is information on the levels of these materials in
the suspended, moving fraction of the solid matrix of water bodies. Flow-through
centrifuges can process large volumes of "whole" water and should be more widely
utilized to obtain samples of this material.
IV. ACCUMULATION
One of the major concerns regarding pesticides in aquatic ecosystems is their accu-
mulation by biota. This process of bioconcentration is defined by Kenaga in Ben-
venue1" as the "amount of pesticide residue accumulated by an organism by adsorp-
tion and by absorption via oral or other route of entry, which results in an increased
concentration of the pesticide by the organism or specific tissues". Other terms often
used in the literature for these processes include biological accumulation, biological
concentration, biological amplification, and environmental magnification. Such proc-
esses are influenced by many properties of the pesticide such as solubility, partitioning,
polarity, and volatility. 115,116
In terms of biological accumulation of DDT, many studies have been performed in
aquatic ecosystems. One of the first major studies was that of DDT partitioning in
biotic components of a Long Island, U.S. estuary."' The water contained only 0.050
pg/l of DDT, while plankton contained 0.04 pg/g or an 800-fold increase in concen-
tration. Invertebrates, plants, and fish further concentrated DDT to 0.08 to l pg/g,
while birds, the highest component of the food chain, contained 1 to 75.5 pg/g or a
concentration factor of up to 1.S1 X 106when compared with water.
Another study of DDT was made in aquatic ecosystems of south Florida by Kolipin-
ski et al."' Waters in the area contained up to 0.03 pg/l of DDT and metabolic prod-
ucts; concentrations in marsh soils, however, were three orders of magnitude higher
and those in biota, including algal mats, crustaceans, and fish, were 3 to 4 orders of
magnitude higher. Sources of these compounds in the area were surface water inflow
and atmospheric inputs as rain contained up to 0.46 pg/l of DDT residues.
Another coastal ecosystem study of DDT and its residues was made by Klaas and
B e l i ~ l e "in
~ a New Jersey salt marsh in 1967 and 1973. Clams contained the highest
DDT residues in 1967 with 9.05 pg/g, but this decreased in 1973 to 0.20 pg/g, a loss
attributed to discontinued use of DDT in 1966. In general, the concentration of DDT
in fauna in the marsh declined by 84 to 99% from 1967 to 1973.
South Carolina estuaries showed accumulation of mirex which had been used for
fire ant control on nearby land.120 Water contained less than 0.01 pg/l mirex, while
sediments contained up t o 70 pg/g. Fish, crabs, and shrimp had mirex levels around 1
pg/g, while birds contained the highest levels of mirex, up to 17.0 pg/g.
Other works showing bioconcentration of organochlorines include DDT in Atlantic
Ocean organisms,12' nonachlor and chlordane in eastern Canadian coastal fauna,122
and DDT and dieldrin in Arctic fur sealslz3and in Oregon estuarine fauna.124
Mechanisms of bioconcentration have been studied. At first such uptake was consid-
ered to act only via food chains in aquatic ecosystems. Plankton accumulated pesti-
cides from water, which were then transferred to invertebrates and fish by direct inges-
tion of the plankton, followed by consumption of such fish and invertebrates by higher
order carnivores such as fish, marine mammals, birds, or humans. Indeed, plankton
do concentrate pesticides directly from water. For example, studies show that generally
levels of pesticides can be higher in organisms consuming plankton than in the plank-
tonic organisms t h e m s e l v e ~ . ~Often ~ ~ . in
~ ~such
~ . ~experiments,
~~ however, algae were
found to contain higher concentrations of pesticides than did organisms higher in the
food hai in.'^'.'^^ Thus, bioaccumulation of pesticides via food chain transfer may not
explain the accumulation phenomenon entirely. Direct uptake of pesticides into the
lipids of biota is another plausible mechanism. Hamelink et al.129rejected the concept
that bioconcentration was a function solely of food chain passage and concluded that
it was a direct exchange between the pesticides in water and body fats. Other studies
with aquatic organisms have also demonstrated that such a direct uptake is
p o ~ s i b l e . ' ~That
~ ~ ~elevated
~' levels in biological tissue may be the result of direct par-
titioning of organics into lipids is also demonstrated by the fact that concentration
factors show a significant positive correlation coefficient with partition coefficients of
organic ~ h e r n i c a 1 s . l ~ ~
V. DEGRADATION
One of the most important aspects of understanding the fate of pesticides in the
environment is a knowledge of degradation mechanisms of the compound in question.
Such degradation processes may lead to the formulation of new chemical compounds
that may be reduced in toxicity to aquatic biota, or in some cases, increased in toxicity.
For example, photooxidation of aldrin produces dieldrin which is of higher toxicity,
while photooxidation of dieldrin or aldrin to photoaldrin or photodieldrin may further
increase t o x i ~ i t y . ~ ~ . ' ~ ~
Degradation of pesticides may occur by either chemical or biological processes or
by a combination of Important chemical reactions include oxidation, re-
duction, hydrolysis, and other nucleophilic reactions and other reactions such as iso-
merization, internal cyclization, and e1iminati0n.l~~ In aquatic environments, biologi-
cal d e g r a d a t i ~ n l ~may
~ , ' ~be~ accomplished by many classes of biota from unicellular
microorganisms to mammals, but bacteria seem to play the most important role. The
sediment component of aquatic ecosystems, especially the sediment-water interface
with its high bacterial biomass, is a major site of biological degradation of pesticides.
Matsumura et isolated bacteria from Lake Michigan bottom silts and found that
93 out of 190 of such isolates could degrade dieldrin to photodieldrin, while only 10
out of 110 water isolates had such a capacity. Patil et al.138isolated bacteria from
various components of a marine ecosystem including seawater, bottom sediments, sur-
face films, algae, and marine plankton; of 100 isolates, 35 were able to degrade DDT
to its products, DDE, TDE(DDD), DDNS, and DDOH. They found marine microor-
ganisms also capable of converting aldrin to dieldrin, photodieldrin, and aldrin diol.
Other areas of pesticide degradation in aquatic ecosystems are wetlands such as
marshes, swamps, and flooded soils. Anaerobic environments are important in such
situations. Sethunathan3$reports rapid degradation of a number of such compounds
under these conditions - lindane, DDT, heptachlor, methoxychlor, endrin, and some
BHC isomers. Slowly degrading compounds included TDE, chlordane, dieldrin, and
aldrin. Important biochemical mechanisms included reductive dechlorination for or-
ganochlorines, favored by higher temperatures and organic matter content, while for
organophosphorus compounds, hydrolysis and nitro group reduction were more com-
mon.
Aquatic organisms such as fish can also degrade pesticides by means of biochemical
processes, resulting in cxidation, hydrolysis, and reduction. Hydrolysis of ester com-
pounds such as organophosphorus compounds, carbamates, pyrethroids, and phen-
oxyacetates is especially important. In mammals, slow metabolism of polycyclic ali-
phatic cyclodienes is possible.
A knowledge of degradation routes may be an important aid in understanding envi-
ronmental likelihood of degradation. Of special importance is the degradation scheme
of DDT summarized in Figure 2 based on studies by K ~ k k e , Fries,140 '~~ Focht,141and
others and summarized in M a t ~ u m u r a . 'Under ~~ oxidative aerobic conditions, DDE
formation is favored, while under reducing conditions, TDE(DDD) is produced.
Glooschenko et al.'" reported that sediments which were anoxic in the upper Great
Lakes tended to have higher amounts of TDE than DDE, while DDE was reported in
planktonic organisms and detritus in the aerobic water column. MacGregors7 studied
DDT and associated degradation products in offshore marine waters in southern Cal-
ifornia and found DDE again typical of fish-dominated aerobic environments while
TDE was associated with discharges from sewers which would be anaerobic. Other
' ~ ~ also shown this trend of DDT - to degrade to DDE or TDE under
s t ~ d e s have
aerobic or anaerobic conditions, respectively.
Pesticides may also degrade in the water column. Edwardss2 has shown that persist-
ence of pesticide compounds in water is a function of several environmental factors
including solubility, temperature, pH, and oxygen conditions. Hill and McCarty14'
found that most organochlorines degrade under anaerobic conditions, except hepta-
chlor epoxide and dieldrin, which are persistent. In terms of ease of degradation, lin-
dane was found to be most degradable followed by heptachlor, endrin, DDT, TDE,
aldrin, heptachlor epoxide, and dieldrin in decreasing order. One of the most thorough
studies of pesticide degradation in water was that of Eichelberger and L i ~ h t e n b e r g , ' ~ ~
who studied the persistence of 28 common compounds in river water over an 8-week
period (12 organochlorine, 9 organophosphorus, and 7 carbamate compounds at an
initial concentration of 10 pg/l). No measurable chemical change was observed for
R-C-R R-CH-R
l OXIDATION I
Cl- C -Cl
t---
Cl-C-Cl DEHYDRO-,
II
l I CHLORINATION -
CI CI
R-C-R
I
OXIDATION
1
R-CH - R
I
DECHLORINATION
DEHYDRO-
R-iH-R
Cl-CH-Cl -CH -cl CHLORINATION CH-CI
2,2- DICHLOROETHANOL 2,2- DICHLOROETHANE 2 - CHLOROETHYLENE

(TDE or DDD)
R-C-R
II
OXIDATION
I DECHLORINATION
0
4,4'- DICHLORO- 2.2-DI(P-CHLOROPHENYL)- 1,l- DI(P-CHLOROPHENYL) -
BENZOPHENONE 1- CHLOROETHANE ETHYLENE
1
*
DECHLORINATION
R-CH-R
I
CH3
-DI(P-CHLORO-
i, ~
1
PHENYL) ETHANE
OXIDATION
R-CH-R R-CH-R
I OXIDATION I
COOH CH20H
2,2-DI(P-CHLORO- 2,2-DI(P-CHLORO-
PHENYL) ACETIC ACID PHENYL) ETHANOL
FIGURE 2. Major microbial degradation pathways of DDT (R=4-chlorophenyl)
BHC, heptachlor epoxide, DDE, DDT, and endrin, while the only stable organophos-
phate was azodrin. The carbamates decreased significantly during a time of 1 week,
and all compounds other than BaygonB completely disappeared over the 8-week pe-
riod. The authors also identified isomers and degradation and chemical conversion
products.
Oloffs et al." investigated the chemical behavior of the compounds DDT, lindane,
a-chlordane, y-chlordane, and TDE, in marine and fresh water. Except for lindane,
the compounds were found to volatilize unless the test vessels were sealed; if this was
done, no metabolic breakdown occurred over a 12-week period. When sediments were
placed into the containers, in addition to water, all residues moved into the sediment
component within 6 weeks. There, most of the lindane was metabolized by microbial
activity, but little change was observed for DDT, TDE, or the chlordanes. Cochrane
et studied the persistence of the propylene glycol butyl ether ester of silvex in
water. After 19 weeks, hydrolysis of this compound to the acid had occurred and it
had been adsorbed onto the sediments.
Degradation studies have also been carried out in natural systems. In one such sys-
tem, the organophosphorus pesticide DursbanC3 was added to fresh water ponds by
Hurlbert et at a concentration of 200 pg/l; after 7 days only 6 pg/l were detected
in the water and most of the compound appeared to have entered the pond sediments.
Biochemical decomposition of this compound produced a toxic oxygen analog which
readily degraded.
Degradation obviously depends upon the nature of the aquatic system investigated.
While not a pesticide considered elsewhere in this volume, a study on diquat illustrates
this well. Simsiman and chest er^'^^ noted that if aquatic weeds were present, 32% of
the diquat was found to break down to water-soluble products after 22 days, while
19% was adsorbed by sediments; if no aquatic plants were present (i.e., less surface
substrate for microbial growth), diquat exhibited very little breakdown even after a
180-day period. Degradation processes, therefore, are influenced by many factors, in-
cluding the amount and type of humic substances present, chemical reactions between
herbicides and organic matter, interactions with sediments, plants, etc. All such factors
must be included in the assessment of both the degradation and the translocation of
such materials.
VI. MODELING
The use of simulation models to describe chemical behavior in aquatic environments

is a technique which holds great promise, since it provides a method of combining
quantitative and qualitative information to obtain a predictive and descriptive scenario
of a pesticide. Such models can be compared with real situations and degradation
pathways or environmental "sinks" can be identified; they may also enable researchers
to predict the environmental dynamics of pesticides and hence the duration of associ-
ated problems. In model-building procedures, postulated mechanisms and associated
models are kept as simple as possible and then gradually made more complex until a
further increase in complexity is not warranted by the data (Occam's razor). The model
which fits the data is not necessarily unique or even the correct one, but it will serve
as a suitable starting point for further investigations and refinement.
In general, the nature of research on pesticides in water focuses attention on what
happens below the air-water interface. This may not be advisable in view of the fact
that accumulation in the surface microlayer has been shown to be ~ubstantial.~' The
"compartments" which should be of interest are, therefore (1) the surface microlayer
of water, (2) subsurface water, (3) suspended solids, living and detrital, (4) aquatic
plants, ( 5 ) fish and (6) sediments. Practically speaking, the construction of a model
requires definition of the "effective sizes" of various compartments, and when this is
done it may be seen that certain compartments may be neglected because they contain
insignificant amounts of the contaminant in question. However, the possibility of
bioaccumulation should not be ignored. It is possible that the significance of the rate
of movement between compartments may outweigh that of the concentrations or quan-
tity within any specific compartments. Some interesting modeling attempts, ranging
from simple pond models to global models, are described below.
Harrison et a1.I4' developed a model to describe the movement of DDT and its break-
down product DDE in an inland ecosystem. The analysis was based on the trophic-
level concept which allowed a simplified quantitative examination of complex "food
web" processes in ecosystems. A mathematical model was derived to indicate the de-
pendence of population size in any trophic level upon the population~in adjacent lev-
els, and to provide a basis for predicting population changes attributable to DDT. The
results suggested that (1) the concentration of DDT in certain species at or near the
top of the trophic structure could continue to rise for many years despite the removal
of DDT inputs to the environment, and (2) an increase in a prey population due to a
decrease in predator numbers could be explosive and the resulting decrease in the prey
food supply could be permanently damaging in that a particular food species could be
eliminated entirely. Robinson130 has noted that some of the conclusions of Harrison
and co-workers were of doubtful validity because of an unrealistic simplifying assump-
tion, namely, that none of the organisms in a trophic level either metabolize or excrete
DDT. This could cast doubt on estimates of the time taken for DDT concentrations
in certain species, at or near the top of the trophic structure, to reach a maximum.
Eberhardt et aI.IZ7used equations of first-order kinetics to model, in some cases
quite accurately, the rate of change of total DDT (the metabolites were not distin-
guished from the parent compound) over a period of 90 days. Their system involved
10 plants, 6 invertebrates, and 2 invertebrate species in a marsh sprayed with DDT.
Woodwell et a1.lZ5have also used first-order equations to obtain estimates of DDT
loads in various global reservoirs as a function of time. The primary reservoirs were
considered t o be the land surface, the atmosphere, the mixed layer of the ocean, and
the abyss. The worldwide pattern of movement of DDT residues appears to be from
the land through the atmosphere into the oceans and thence to the oceanic abyss. Their
calculations, based on the fragmentary data available on rates of movement and sizes
of various pools of DDT residues, led to the conclusion that concentrations in the
atmosphere and in the mixed layer of the oceans lag by only a few years behind the
amounts of DDT produced and used annually throughout the world. Their model sug-
gested that the maximum concentrations of DDT residues in air occurred in 1966 and
would occur in the mixed layer of the oceans in 1971.
In another aquatic model, Neely et al.14" have developed a mathematical treatment
which is capable of predicting concentration-time profiles in a river downstream from
a spill of water-soluble or partially soluble chemical. The river is visualized as a series
of contiguous, stirred-flow compartments in which the output from each compartment
is fed into the next compartment, and volatilization (assumed to follow first-order
kinetics) is the only other output process considered. The series of differential equa-
tions resulting from a consideration of n compartments was solved by computer and
modeled fairly well the concentration-time profiles of chloroform at two points in the
Mississippi River downstream from a major chloroform spill. The model is also capa-
ble of handling situations in which the chemical enters the water at a constant (low)
rate over a fixed interval of time.
Neely and developed a three-compartment model involving water, sediment,
and fish which simulated adequately the distribution and persistence of chlorpyrifos
(O,Odiethyl-0[3,5,6-trichloro-2-pyridyl]phosphorothioate)in small experimental
ponds. Their work indicates the extreme care that must be exercised in interpreting
bioconcentration ratios from field observations. Without any prior knowledge of the
initial conditions or the chemical and physical properties of the pesticide under study,
it is impossible to decide whether a steady state has been reached so that a general
bioconcentration factor can be defined.
Marshal1 and RobertslS0 have also developed a three-compartment model, in this
case, water, suspended solids and sediment, to simulate the distribution and persistence
of fenitrothion (0,Odimethyl-O[pnitro-m-tolyl]phosphorothionate) in ponds. Proc-
esses considered most likely to explain the rapid disappearance of fenitrothion from
the water phase included adsorption on sediments, photolysis, and microbial degrada-
tion, but not hydrolysis or volatilization. There were not sufficient data with which to
test rigorously their hypothesis; however, the authors were able to identify specifically
areas requiring further investigation - photolysis (quantum yields, effects of oxygen,
role of natural sensitizers and quenchers), microbial degradation (kinetics in water and
sediments), sediment sorption-desorption (in particular, definition of the volume of
the adsorptive phase), and such system-specific parameters as water volume, mean
depth, size and effective thickness of sediments, concentration of suspended solids,
and bacterial densities in water and sediments.
VII. TOXICOLOGY
The mode of action of various classes of pesticides on target pests has been exten-
sively r e v i e ~ e d ' and
~ ~ will
~ ~ not
~ ~be~ described
' ~ ~ here. The concern of environmental
agencies, however, is usually more with the effects of pesticides on nontarget organ-
isms; it is frequently the task of such agencies to identify the cause(s) of an observed
effect as well as to determine the effect of known chemical compounds. These effects
on nontarget organisms have recently received a good deal of a t t e n t i ~ n . ' ~ ~ - ' ~ '
During the past 10 years it has become apparent that environmental risk assessments
cannot be based solely on acute toxicity data: there is a range of subtle sublethal or
chronic effects on aquatic organisms which may be associated with low doses of pesti-
cides ranging from behavioral aberrations to mutagenesis, teratogenesis, and carcino-
genesis, or associated with accumulation of residues to levels undesirable to the con-
sumer. In addition, there may be synergistic or antagonistic effects of two or more
xenobiotics. The need to be able to identify even smaller amounts of toxic compounds
(and pesticides in particular) in water and tissues of aquatic organisms in order to be
able to identify sublethal cause-effect linkages places great demands on the skill and
ingenuity of the analytical chemist.
It should also be borne in mind that environmental risk assessments of pesticides
are not determined only by toxicity data (acute or sublethal/chronic), but should take
into account the amount of the material in use and its patterns of use and disposal, its
chemical and physical properties, and its pathways and extent of environmental deg-
radation - in other words - its exposure. A toxic compound which has a very low
persistence in aquatic ecosystems is unlikely to represent a major danger unless it is
introduced continuously. In this regard, the use of reliable models which take into
account both the environmental concentrations and the rates of significant processes
is essential.
VIII. MONITORING
With the introduction of hundreds of new substances, some of which are pesticides,
into the commercial market each year, there is an increased likelihood of finding some
of them, or their transformation products, persisting in the aquatic environment. The
international move t o pre-market testing will do much toward ensuring some control
of this, but despite such efforts, compounds will become dispersed in the environment
and some of them will exert undesirable effects on the ecosystem. Much diligence will
be required to detect these substances at any early stage, before the consequences of
their release become disastrous. These investigations should have as their objective the
establishment of the occurrence, the rates of processes affecting persistence, and the
toxic effects of chemicals on organisms located in various parts of the ecosystem.
Armed with such information, the design of a sound monitoring program should lead
to reliable answers about these materials when found in the environment.
Also required for monitoring will be the development of analytical techniques in
order t o process the range of samples - surface waters, sediments, biological tissues
of many sorts, air, soil, groundwater, etc. - that will be collected. No specific com-
ments can be made which will be applicable for the variety of extraction, concentra-
tion, isolation (from interferences), and detecting procedures which will be needed.
These developments will have t o be both general, for investigative monitoring, and
compound specific, for routine surveillance. The surveillance programs which will be
required must be founded on a sound data base of rates and identified critical compo-
nents in the ecosystem. Together with such information, surveillance data on environ-
mental levels should be incorporated into practical models designed to answer the ques-
tions of where, how much, and how long.
REFERENCES
1. Fowler, H. W. and Fowler, F. G . , The Concise Oxford Dictionary of Current English, 4th ed.,
revised by Mclntosh, E., Oxford University Press, Oxford, 1951.
2. Tu, C. M., Miles, J. R. W. and Harris, C. R., Soil microbial degradation of aldrin, Life Sci., 7,
311,1968.
3. Hemmett, R. B., Jr. and Faust, S. D., Biodegradation kinetics of 2,4-dichlorophenoxyaceticacid by
aquatic organisms, Res. Sci., 29, 191,1968.
4. Konard, J. G.,Chesters, G. and Armstrong, D. E., Soil degradation of malathion, a phosphorodi-
thioate insecticide, Proc. SoilSci. Soc. Am., 33,259, 1969.
5. Kenaga, E. E., Partitioning and uptake of pesticides in biological systems, in Proc. Transp. Persist.
Chem. Conf., National Research Council, Ottawa, 1974,19.
6. Macek, K. J., Biological magnification of pesticide residues in food chains, Proc. Symp. Biol. Im-
pact. Pest. Environ., Gillett, J.W., Ed., Oregon State University, Corvallis, 1970, 17.
7. Norstrom, R. J., Hallett, D. J. and Sonstegard, R. A., Coho salmon (Oncorhynchus kisutch) and
herring gulls (Larus argentus) as indicators of organochlorine contamination in Lake Ontario, J. Fish
Res. Board Can., 35, 1401,1978.
8. Oda, J. and Muller, W., Identification of a mammalian breakdown product of dieldrin, in Environ-
mental Quality and Safety, Vol. 1, Coulston, F. and Korte, F., Eds., Academic Press, New York,
1972,248.
9. Khan, M. A. Q., Korte, F., and Payne, J. F. Metabolism of pesticides by aquatic animals, in Pesti-
cidesin AquaticEnvironments, Khan, M. A. Q . , Ed., Plenum Press, New York, 1977,191.
10. Hathaway, D. E., Biotransformations, in Foreign Compound Metabolism in Mammals, Vol. 1,
Chem. Soc. Specialist Periodical Report, Hathaway, D. E., Brown, S. S., Chasseaud, and Hutson,
D. H., Eds., Chemical Society, London, 1970.
11. Oloffs, P. C., Albright, L. J. and Szeto, S. Y., Fate and behaviour of five chlorinated hydrocarbons
in three natural waters, Can. J. Microbiol., 18, 1393,1972.
12. Goring, C. A. I., Laskowski, D. A., Hamaker, J. W., and Meikle, R. W., Principles of pesticide
degradation in soil, in Environmental Dynamics of Pesticides, Haque, R. and Freed, V. H., Eds.,
American Chemical Society, Washington, D.C., 1975,135.
13. Freeman, H.P., Taylor, A. W., and Edwards, W. M., Heptachlor and dieldrin disappearance from
a field soil measured by annual residue determinations, J. Agric. Food Chem., 23, 1101, 1975.
14. Freal, J. J., and Chadwick, R. W., Metabolism of hexachlorocyclohexane to chlorophenols and
effect of isomer pretreatment o n lindane metabolism in rat, J. Agric. Food Chem., 21,424,1973.
15. Brooks, G. T.,Degradation of organochlorine insecticides, problems and possibilities, in Proc. 2nd
I U P A C Congress of Pesticide Chemistry, Fate of Pesticides in the Environment, Vol. 6,Tahori, A.
S., Ed., Cordon and Breach, 1972.
16. Reed, W. T. and Forgash, A. J., Lindane: metabolism to a new isomer of pentachlorocyclohexane,
Science, 160, 1232, 1968.
17. Brooks, G. T., Perspectives in cyclodiene metabolism, in Proc. Symp. Science and Technology of
Residuaj Insecticides in Food Production with Special Reference to Aldrin and Dieldrin, Shell Chem-
ical Co., New York, 1968,89.
18. Brooks, G. T., Action of chlorinated insecticides, in Chlorinated Insecticides, Vol. 2, CRC Press,
Boca Raton, Fla., 1974,chap. 3.
19. Weisberger, I., Detera, S., and Klein, W., Ecological chemistry. LXXXVIII. Isolation and identifi-
cation of some heptachlor - "C metabolites from plants and soil, Chemosphere, 3,221,1974.
20. Miles, J. R. W., Tu, C. M., and Harris, C. R., Metabolism of heptachlor and its degradation prod-
ucts by soil microorganisms, J . Econ. Entomol.,62, 1334, 1969.
21. Georgacakis, E. and Khan, M. A. Q., Toxicity of the photoisomers of cyclodiene insecticides to
freshwater animals, Nature (London), 233, 120, 1971.
22. Sandi, E., Reduction of parathion induced by light, Nature (London), 181,499, 1958.
23. Nakatsugawa, T. and Dahm, P. A., Microsomal metabolism of parathion, Biochem. Pharmacol.,
16.25, 1967.
24. Potter, J . L. and O'Brien, R. D., Parathion activation by livers of aquatic and terrestrial vertebrates,
Science, 144, 55, 1964.
25. Graetz, D. A., Chesters, G., Daniel, T. C., Newland, L. W., and Lee, G. B., J . Water Pollut. Cont.
Fed.,42, R76, 1970.
26. Miyamoto, J., Degradation of fenitrothion in terrestrial and aquatic environments including photo-
lytic and microbial reactions, in Proc. Symp. on Fenitrothion: The Long Term Effects of Its Use in
Forest Ecosystems, Report 16073, Roberts, J. R., Greenhalgh, R., and Marshall, W. K., Eds. Na-
tional Research Council of Canada, Ottawa, 1977.
27. Miyamoto, J. and Mikami, N., Photodecomposition of sumithion in water and on soil surface, un-
published report; cited in Proc. of Symp. on Fenitrothion: The Long Term Effects of Its Use in
Forest Ecosystems., Report 16073, Roberts, J. R., Greenhalgh, R., and Marshall, W. K., Eds., Na-
tional Research Council of Canada, 1977.
28. Eaton, J. G., Chronic malathion toxicity to the bluegill (Lepomis macrochjrus), Water Res., 4, 673,
1970.
29. Weiss, C. M. and Gakstatter, J. H., Detection of pesticides in water by biochemical assay, J. Water
Pollut. Cont. Fed., 36, 240, 1964.
30. Zuckerman, B. M., Deubert, K., Mackiewicz, M., and Gunner, H., Studies on the biodegradation
of parathion, Plant and Soil, 33,273, 1970.
3 1. Sethunathan, N., Microbial degradation of insecticides in flooded soil and in anaerobic cultures, Res.
Rev.,47, 143, 1973.
32. Miller, C. W., Zuckerman, B. M., and Charig, A. J., Water translocation of diazinon-C" and par-
~ a model cranberry bog and subsequent occurrence in fish and mussels, Trans. Am.
a t h i ~ n - S 'off
Fish. Soc., 95,345, 1966.
33. Aly, 0. M. and Faust, S. D., Studies on the fate of 2.4-D and ester derivatives in natural surface
waters, J . Agric. Food Chem., 12, 541, 1964.
34. Zepp, R. G., Wolfe, N. L., Gordon, J. A., and Baughman, G. L., Dynamics of 2,4-D esters in
surface waters. Hydrolysis, photolysis and vaporization, Environ. Sci. Technol., 9, 1144, 1975.
35. Loos, M. A., Phenoxyalkanoic acids, in Herbicides: Chemistry, Degradation and Mode of Action,
2nd ed., Kearney, P. C. and Kaufman, D. D. Eds., Marcel Dekker, New York, 1976, chap. I.
36. Pionke, H. B. and Chesters, G., Pesticide-sediment-water interactions, J . Environ. Qual., 2, 29,
1973.
37. Faust, S. D., Pollution of the water environment by organic pesticides, Clin. Pharmacol. Ther., 5,
677, 1964.
38. Buser, H. R., Polychlorinated dibenzo-p-dioxins. Separation and identification of isomers by gas
chromatography-mass spectrometry, J. Chromatogr., 114,95, 1975.
39. Jensen, S. and Renberg, L., Contaminants in pentachlorophenol: chlorinated dioxins and predioxins,
Ambio, 1.62, 1972.
40. Miller, R. A., Norris, L. A., and Hawkes, C. L., Toxicity of TCDD in aquatic organisms, Environ.
Health Perspect., 5, 177, 1973.
41. Kearney, P. C., Woolson, E. A., and Ellington, C. P., Jr., Persistence and metabolism of chloro-
dioxins in soils, Environ. Sci. and Technol.,6, 1017, 1972.
42. Smith, J. W. and Sheets, T. J., Uptake, distribution and metabolism of monuron and diuron by
several plants, J . Agric. Food Chem., 15, 577, 1967.
43. Dalton, R. L., Evans, A. W., and Rhodes, R. C., Disappearance of diuron from cotton field soils,
Weeds, 14, 3 1, 1966.
44. Armstrong, D. E.. Chesters, G., and Harris, R. F., Atrazine hydrolysis in soil, in Proc. Soil Sci.
Soc. Am., 31, 1967,61.
45. Skipper, H. D., Gilmour, C. M., and Furtick, W. R., Microbial versus chemical degradation of
atrazine in soils, in Proc. Soil Sci. Sco. Am., 3 1, 1967, 653.
46. Pape, B. E. and Zabik, M. J., Photochemistry of selected 2-chloro- and 2-methythio-4,6-(dialkylam-
ino)-S-triazineherbicides, J. Agric. Food Chem., 18, 202, 1970.
47. Benyon, K. I., Bosio, P., and Elgar, K. E., The analysis of crops and soils for the triazine herbicide
cyanazine and some of its degradation products. 11. Results, Pest. Sci., 3,401, 1972.
48. Muir, D. C. and Baker, B. E., Detection of triazine herbicides and their degradation products in tile-
drain water from fields under intensive corn (maize) production, J . Agric. Food Chem., 24, 122,
1976.
49. Kaufman, D. D. and Kearney, P. C., Microbial degradation of S-triazine herbicides, Res. Rev., 32,
235, 1970.
50. Zimdahl, R. L., Freed, V. H., Montgomery, M. L., and Furtick, W. R.,The degradation of triazine
and macil herbicides in soil, Weed Res., 10, 18, 1970.
51. Edwards, C. A., Persistent Pesticides in the Environment, 2nd ed., CRC Press, Boca Raton, Fla.,
1973.
52. Edwards, C. A., Pesticide residues in soil and water, in Environmental Pollution by Pesticides, Ed-
wards, C. A., Ed., Plenum Press, New York, 1973,409.
53. Glotfelty, D. E. and Caro, J. H., Introduction, transport and fate of persistent pesticides in the
atmosphere, in Proc. Symp. on Removal of Trace Contaminants from the Air, ACS Symp. Ser., 17,
1975,42.
54. Bergsteinsson, J. T. and Baier, W., Meteorological Aspects of Pollution in Relation to Agricultural
Pesticides, Canada Committee of Agricultural Meteorology, Research Branch, Canadian Department
of Agriculture, Ottawa, 1971.
55. Wheatley, G. A. Pesticides in the atmosphere, in Environmental Pollution by Pesticides, Edwards,
C. A., Ed., Plenum Press, New York, 1973,366.
56. Makhon'Ko, K. P., Simplified theoretical notion of contaminant removal by precipitation from the
atmosphere, Tellus, 19,467, 1967.
57. Hage, K. D., Microscale and mesoscale transport, in Meteorological Aspects of Pollution in Relation
to Agricultural Practices, Bergsteinssen, J . T. and Baier, W., Eds., Canada Committee of Agricul-
tural Meteorology, Research Branch, Canadian Department of Agriculture, Ottawa, 1971,25.
58. Spencer, W. F., Distribution of pesticides between soil, water, and air, in Proc. Int. Symp. Pest.
Soil, Michigan State University, East Lansing, 1970, 120.
59. Lloyd-Jones, C. P., Evaporation of DDT, Nature(London), 229, 65, 1971.
60. Risebrough, R. W., Huggett, R. J., Griffin, J. J., and Goldberg, E. D., Pesticides: transatlantic
movements in the northeast trades, Science, 159, 1233, 1968.
61. Cohen, J. M. and Pinkerton, C., Widespread translocation of pesticides by air transport and rain-
out, Adv. Chem., 163, 1966.
62. Peakall, D. B., DDT in rainwater in New York following applications in the Pacific northwest,
Atmos. Environ., 10,899, 1976.
63. Stanley, C. W., Barney, J. E., 11, Helton, M. R., and Yobs, A. R., Measurement of atmospheric
levels of pesticides, Environ. Sci. Technol., 5, 430, 1971.
64. Strachan, W. M. J., Huneault, H., Schertzer, W. M., and Elder, F. C., Organochlorines in precipi-
tation in the Great Lakes region, in Proc. Int. Symp. Anal. Hydrocarbons, Halogenated Hydro-
Carbons, Hamilton, Afghan, B.K. and MacKay, D., Eds., Plenum Press, New York, 1980,387.
65. Sodergren, A., Transport, distribution, and degradation of DDT and PCB in a south Swedish lake
ecosystem, Vatten, 2, 90, 1973.
66. Benvenue, A., Ogata, J. N., and Hylin, J. W., Organochlorine pesticides in rainwater, Oahu, Hawaii,
Bull. Environ. Cont. Toxic., 8, 238, 1972.
67. Masahiro, 0. and Takahisa, H. Alpha- ad gamma-BHC in Tokyo rainwater (December 1968 to
November 1969), Environ. Pollut., 9,283, 1975.
68. McClure, V. E. and Lagrange, J., Deposition of heavy chlorinated hydrocarbons from the atmos-
phere, BUN.Environ. Cont. Toxic., 17,219, 1977.
69. Tarrant, K. R. and Tatton, J. O'G., Organochlorine pesticides in rainwater in the British Isles, Nature
(London), 219, 725, 1968.
70. Edwards, C. A., Nature and origins of pollution of aquatic systems by pesticides, in Pesticides in
Aquatic Environments, Khan, M. A. Q., Ed., Plenum Press, New York, 1977, 11.
71. Seba, D. B. and Corcoran, E. F., Surface slicks as concentrators of pesticides in the marine environ-
ment, Pest. Monit. J., 3, 190, 1969.
72. Wershaw, R. L., Burcar, P. J., and Goldberg, M. C., Interaction of pesticides with natural organic
material, Environ. Sci. Technol., 3, 271, 1969.
73. Odum, W. E., Woodwell, G. M., and Wurster, C. F., DDT residues absorbed from organic detritus
by fiddler crabs, Science, 164, 576, 1969.
74. Pierce, R. H., Jr., Olney, C. E., and Felbeck, G. T., Jr., pp'-DDT adsorption to suspended particu-
late matter in sea water, Geochim. Cosmochim. Acta, 38, 1061, 1974.
75. Cox, J. L., DDT residues in rnarinephytoplankton, Res. Rev.,44,23, 1972.
76. Lichtenberg, J. J., Eichelberger, J. W., Dressman, R. C., and Longbottom, J. E., Pesticides in
surface waters of the United States - a 5-year summary, 1964-68, Pest. Monit. J.,4, 71, 1970.
77. Zabik, M. J., Pape, B. E., and Bedford, J. W., Effect of urban and agricultural pesticide use on
residue levels in the Red Cedar River, Pest. Monit. J., 5, 1971.
78. Truhlar, J. E. and Reed, L. A., Occurrence of pesticide residues in four streams draining different
land-use areas in Pennsylvania, 1969-71, Pest. Monit. J., 10, 101, 1976.
7 9 . Schulze, J. A., Manigold, D. B., and Andrews, F. L., Pesticides in selected western streams, 1968-
7 1 , Pest. Monit. J., 7 , 7 3 , 1973.
80. Richard, J. J., Junk, G. A., Avery, M. J., Nehring, N. L., Fritz, J. S., and Svec, H. J., Analysis of
various Iowa waters for selected pesticides: atrazine, DDE, and dieldrin - 1974, Pest. Monit. J.,
9(3), 117, 1975.
81. Ginn, T. M. and Fisher, F. M., Jr., Studies on the distribution and flux of pesticides in waterways
associated with a rice field-marshland ecosystem, Pest. Monit. J., 8, 23, 1974.
82. Gummer, W. D., Pesticide monitoring in the Prairies of western Canada, Water Quality Branch,
Inland Waters Directorate, Western and Northern Region, unpublished, 1978.
83. Harris, C. R . and Miles, J. R. W., Pesticide residues in the Great Lakes region of Canada, Res.
Rev., 5 7 . 2 7 , 1975.
84. Schacht, R. A., Pesticides in the Illinois waters of Lake Michigan, Rep. EPA 660/3-74-002, Office
Research Development, U.S. Environmental Protection Agency, Washington, D.C., 1974.
85. Miles, J . R . W., Bolton, E. F., and Harris, C. R., Insecticide and nutrient transport in water related
to agricultural land use of a stream basin in Ontario, Canada, Arch. Environ. Cont. Toxicol., 5 ,
119, 1976.
86. Glooschenko, W. A., Strachan, W. M. J., and Sampson, R. C. J., Distribution of pesticides and
polychlorinated biphenyls in water, sediment and seston of the upper Great Lakes - 1974. Pest.
Monit. J . , 10, 6 1 , 1976.
87. MacGregor, J. S., Changes in the amount and proportions of DDT and its metabolites, DDE and
DDD, in the marine environment off southern California, 1949-72, Fish. Bull., 7 2 , 275, 1974.
88. Mattraw, H . C., Jr., Occurrence of chlorinated hydrocarbon insecticides, southern Florida - 1968-
7 2 , Pest. Monit. J., 9 , 106, 1975.
89. Schafer, M. L., Peeler, J. T., Gardner, W. S., and Campbell, J. E., Pesticides in drinking water
from the Mississippi and Missouri Rivers, Environ. Sci. Technol., 3(12), 1261, 1968.
90. Robeck, G. G., Dostal, K. A., Cohen, J. M., and Kreissl, J. F., Effectiveness of water treatment
processes in pesticide removal, J. Am. Water Works Assoc., 57, 181, 1965.
91. Kaiser, K. L. E., Mirex: an unrecognized contaminant of fishes from Lake Ontario, Science, 185,
523, 1974.
92. Holdrinet, M., Frank, R., Thomas, R. L., and Hetling, L., Mirex in sediments of Lake Ontario, J.
Great Lakes Res., 4 , 6 9 , 1978.
93. Greve, P. A. and Wit, S. L., Endosulfan in the Rhine River, J. Water Pollut. Cont. Fed., 43, 2338,
1971.
94. Greve, P. A. Potentially hazardous substances in surface waters. I. Pesticides in the river Rhine.,
Sci. Total Envir., 1 173, 1972.
95. Greichus, Y . A., Greichus, A., Ammann, B. D., and Hopcraft, J., Insecticides, polychlorinated
biphenyls and metals in African lake ecosystems. 111. Lake Nakuru, Kenya, Bull. Environ. Cont.
Toxicol., 19, 454, 1978.
96. Reinold, R. J., Chlorinated hydrocarbon pesticides and mercury in coastal biota, Puerto Rico and
the U.S. Virgin Islands, 1972-74, Pest. Monit. J . , 9 , 39, 1975.
97. Adams, R . S., Jr., Factors influencing soil adsorption and bioactivity of pesticides, Res. Rev., 47,
1 , 1973.
98. Lotse, E. G., Graetz. D. A., Chesters, G., Lee, B. G., and Newland, L. W., Lindane absorption by
lake sediments, Environ. Sci. Technol., 2, 353, 1968.
99. Huang, J-C., Effect of selected factors on pesticide sorption and desorption in the aquatic system,
J. Water Pollut. Cont. Fed., 4 3 , 1739, 1971.
100. Boucher, F. R. and Lee, G. F., Adsorption of lindane and dieldrin pesticides on unconsolidated
aquifer sands, Environ. Sci. Technol., 6 , 538, 1972.
101. Wang, W. -C., Lee, G. F., and Spyridakis, D., Adsorption of parathion in a multicomponent solu-
tion, Water Res., 6 , 1219, 1972.
102. Leshniowsky, W. O., Dugan, P. R., Pfister, R. M., Frea, J. J., and Randles, C. I., Adsorption of
chlorinated hydrocarbon pesticides by microbial floc and lake sediment and its ecological implica-
tions., in Proc. 13th Conf. Great Lakes Res., Ann Arbor, Mich., 1970,611.
103. Wiersma, G. B . , Tai, H., and Sand, P. F., Pesticide residues in soil from eight cities - 1969, Pest.
Monit. J . , 6 , 126, 1972.
104. Carey, A. E . , Wiersma, G. B., and Tai, H., Pesticide residues in urban soils from 14 United States
cities, 1970, Pest. Monit. J., 10, 54, 1976.
105. Miles. J. R. W. and Harris, C. R. Insecticide residues in a stream and a controlled drainage system
in agricultural areas of southwestern Ontario, 1970, Pest. Monit. J., 5 , 89, 1971.
106. Miles, J. R. W., Insecticide residues on stream sediment in Ontario, Canada, Pest. Monit. J., 10,
87, 1976.
107. Frank, R., Armstrong, A. E.. Boelens, R. G., Braun, H. E., and Douglas, C. W., Organochlorine
insecticide residues in sediment and fish tissues, Ontario, Canada, Pest. Monit. J., 7 , 165, 1974.
108. Glooschenko, W. A., Capobianco, J., Coburn, J., and Glooschenko, V., Geochemical studies in a
suburban marsh with reference to metal and organochlorine contaminants, unpublished Rep., Can-
ada Centre Inland Waters, Burlington, Ontario, 1978.
109. Frank, R., Thomas, R. L., Holdrinet, M., Kemp, A. L. W., and Braun, H. E., Organochlorine
pesticides and PCB in surficial sediments (1968) and sediment cores (1976) from Lake Ontario, J.
Great Lakes Res., 5, 18, 1979.
110. Frank, R., Holdrinet, M., Braun, H.E., Thomas, R. L., Kemp, A. L. W., and Jaquet, J.-M.,
Organochlorine insecticides and PCBs in sediments of Lake St. Clair (1970 and 1974) and Lake Erie
(1971). Sci. Total Environ., 8, 205, 1977.
111. Hom, W., Risebrough, R. W., Soutar, A., and Young, D. R., Deposition of DDE and polychlroin-
ated biphenyls in dated sediments of the Santa Barbara basin, Science, 184, 1197, 1974.
112. Law, L. M. and Goerlitz, D. F., Selected chlorinated hydrocarbons in bottom material from streams
tributary to San Francisco Bay, Pest. Monit. J., 8, 33, 1974.
113. Hall, K. J., Yesaki, I., and Chan, J., Trace metals and chlorinated hydrocarbons in the sediments
of a metropolitan watershed, Tech. Rep. 10, Westwater Research Centre, University of British Col-
umbia, Vancouver, 1976.
114. Cannone, P. and Mamarbachi, G., Residus des insecticides organochlores dans les sediments du haut
estuairedu fleuve St. -Laurent., Bull. Environ. Cont. Toxicol., 14, 83, 1975.
115. Benvenue, A., The "bioconcentration" aspects of DDT in the environment, Res. Rev., 61, 37, 1976.
116. Kenaga, E. E., Partitioning and uptake of pesticides in biological systems, Environ. Sci. Res., 6,
217, 1975.
117. Woodwell, G. M., Wurster, C. F., Jr., and Isaacson, P. A., DDT residues in an east coast estuary:
a case of biological concentration of a persistent insecticide, Science, 156, 821, 1967.
118. Kolipinski, M. C., Higer, A. L., and Yates, M. L., Organochlorine insecticide residues in Everglades
National Park and Loxahatchee National Wildlife Refuge, Florida, Pest. Monit. J., 5, 281, 1971.
119. Klaas, E. E. and Belisle, A. A. Organochlorine pesticide and polychlorinated biphenyl residues in
selected fauna from a New Jersey salt marsh - 1967 vs. 1973, Pest. Monit. J., 10, 149, 1977.
120. Borthwick, P. W., Duke, T. W., Wilson, A. J., Jr., Lowe, J . I., Patrick, J . M., Jr., and Oberhen,
J . C., Accumulation and movement of mirex in selected estuaries of South Carolina, 1967-71, Pest.
Monit. J., 7 , 6 , 1973.
121. Harvey, G . R., Miklas, H . P., Bowen, V. T., and Steinhauer, W. G., Observations on the distribution
of chlorinated hydrocarbons in Atlantic Ocean organisms., J. Mar. Res., 32, 103, 1974.
122. Zitko, V., Nonachlor and chlordane in aquatic fauna, Chemosphere, 7, 3, 1978.
123. Kurtz, D. A. and Kim, K. C., Chlorinated hydrocarbon and PCB residues in tissues and lice of
Northern Fur Seals, Pest. Monit. J., 10, 79, 1976.
124. Claeys, R. R., Caldwell, R. S., Cutshall, N. H., and Holton, R., Chlorinated pesticides and poly-
chlorinated biphenyls in marine species, Oregon/Washington Coast, 1972, Pest. Monit. J.,9, 2, 1975.
125. Woodwell, G . M., Craig, P. P., and Johnson, H. A., DDT in the Biosphere: where does it go?,
Science, 174, 1101, 1971.
126. Petrocelli, S. R., The interactions of a chlorinated hydrocarbon insecticide among the water, sedi-
ments and biota in an estuarine system, Diss. Abstr. 834, 1974, 3109, Ph.D. Thesis, Texas A&M
University, College Station, 1973.
127. Eberhardt, L. L., Meeks, R. L., and Peterle, T. J., Food chain model for DDT kinetics in a fresh-
water marsh, Nature (London), 230, 60, 1971.
128. Moore, R., Toro, E., Stanton, M., and Khan, M. A. Q., Absorption and elimination of I4C-alpha-
and gamma-chlordane by a freshwater alga, daphnid, and goldfish, Arch. Environ. Cont. Toxicol.,
6, 41 1, 1977.
129. Hamelink, J . L., Waybrant, R. C., and Ball, R. C., A proposal: exchange equilibria control the
degree chlorinated hydrocarbons are biologically magnified in lentic environments, Trans. Am. Fish.
Soc., 100,207, 1971.
130. Robinson, J., Dynamics of pesticide residues in the environment, in Environmental Pollution by
Pesticides, Edwards, C . A., Ed., Plenum Press, London, 1973.
131. Harding, G . C. H. and Vass, W. P., Uptake from sea water and clearance of "C - p,p' - DDT by
the marine copepod Calanus finmarchicus, J. Fish. Res. Bd. Can., 34, 177, 1977.
132. Neely, W. B., Branson, D. R., and Blau, G . E., Partition coefficient to measure bioconcentration
potential of organic chemicals in fish, Environ. Sci. Technol., 8, 1113, 1974.
133. Haque, R., Kearney, P. C., and Freed, V. H., Dynamics of pesticides in aquatic environments, in
Pesticidesin AquaticEnvironments, Khan, M. A . Q., Ed., Plenum Press, New York, 1977, 39.
134. Matsumura, F., Degradation of pesticide residues in the environment, in Environmental Pollution
by Pesticides, Edwards, C . A., Ed., Plenum Press, London, 1973,494.
135. Paris, D. F. and Lewis, D. L., Chemical and microbial degradation of ten selected pesticides in
aquatic systems, Res. Rev., 45, 95, 1973.
136. Crosby, D. G., The fate of pesticides in the environment, Annu. Rev. Plant Physiol., 24,467, 1973.
137. Matsumura, F., Patil, K. C., and Boush, G. M., Formation of "photodieldrin" by microorganisms,
Science, 170, 1206, 1970.
138. Patil, K. C., Matsumura, F., and Boush, G. M., Metabolic transformation of DDT, dieldrin, aldrin,
and endrin by marine microorganisms, Environ. Sci. Technol, 6,629, 1972.
139. Kokke, R., DDT: its action and degradation in bacterial populations, Nature (London), 226, 972,
1970.
140. Fries, G. F., Degradation of chlorinated hydrocarbons under anaerobic conditions, Adv. Chem. Sci.,
111,256, 1972.
141. Focht, D. D., Microbial degradation of DDT nletabolites to carbon dioxide, water, and chloride,
Bull. Environ. Cont. Toxicol.,7,52, 1972.
142. Hill, D. W. and McCarty, P. C., Anaerobic degradation of selected chlorinated hydrocarbon pesti-
cides, 3. Water Pollut. Cont. Fed., 39, 1259, 1967.
143. Eichelberger, J. W. and Lichtenberg, J. J., Persistence of pesticides in river water, Environ. Sci.
Technol.,5, 541, 1971.
144. Cochrane, D. R., Pope, J. D., Jr., Nicholson, H. P., and Bailey, G. W., The persistence of silvex in
water and hydrosoil, Water Resour. Res., 3,517, 1967.
145. Hurlbert, S. H., Mulla, M. S., Keith, J. O., Westlake, W. E., and Dusch, M. E., Biological effects
and persistence of dursban in freshwater ponds, J. Econ. Entomol., 63,43, 1970.
146. Simsiman, G. V. and Chesters, G., Persistence of diquat in the aquatic environment, Water Res.,
10, 105, 1976.
147. Harrison, H. L., Loucks, 0.L., Mitchell, J. W., Parkhurst, D. F., Tracy, C. R., Watts, D. G., and
Yannacone, V. J., Jr., Systems studies of DDT transport. A systems analysis provides new insights
for predicting long-term impacts of DDT in ecosystems, Science, 170, 503, 1970.
148. Neely, W. B., Blau, G. E., and Alfrey, T., Jr., Mathematical models predict concentration - time
profiles resulting from chemical spill in a river, Environ. Sci. Technol., 10,72, 1976.
149. Neely, W. B. and Blau, G. E., The use of laboratory data to predict the distribution of chlorpyrifos
in a fish pond, in Pesticides in Aquatic Environments, Khan, M. A. Q., Ed., Plenum Press, New
York, 1977,145.
150. Marshall, W. K. and Roberts, J. R., Simulation modeling of the distribution of pesticides in ponds,
in Proc. Symp. Fenitrothion: The Long-Term Effects of Its Use in Forest Ecosystems, NRCC No.
16073, National Research Council of Canada, Ottawa, Ontario, 1977, 253.
151. Kohn, G. K., Ed., Mechanism of Pesticide Action, Am. Chem. Soc. Symp. Ser. 2, American Chem-
ical Society, Washington, D.C., 1974.
152. Matsumura F., Toxicology of Insecticides, Plenum Press, New York, 1975.
153. O'Brien, R. D., Insecticides: Action and Metabolism, Academic Press, New York, 1970.
154. Khan, M. A. Q. and Bederka, J. P., Jr., Eds., Survival in Toxic Environments, Academic Press,
New York, 1974.
155. Veith, G. D. and Konasewich, D. E., Eds., Structure-Activity Correlations in Studies of Toxicity
and Bioconcentration with Aquatic Organisms, Proc. Symp. at Canada Centre for Inland Waters,
Burlington, Ontario, 1975, publ. by International Joint Commission, Great Lakes Research Advisory
Board, Windsor, Ont.
156. Mayer, F. L. and Hamelink, J. L., Eds., Aquatic Toxicology and Hazard Evaluation, Proc. First
Ann. Symp. Aq. Toxicol., Memphis, Tenn., 1976; American Society for Testing and Materials, Phil-
adelphia, 1977.
157. Koeman, J. H. and Strik, J. J. T. W. A., Eds., Sublethal Effects of Toxic Chemicals on Aqua tic
Animals, Proc. Swedish-Netherlands Symp., Wageningen, The Netherland, Sept. 1975. Elsevier, New
York, 1975.
Chapter 2
BASIC PRINCIPLES AND PRACTICES ON THE ANALYSIS OF

PESTICIDES
Alfred S . Y . Chau and Hing-Biu Lee
TABLE OF CONTENTS
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .27
I1. General Sequence for Pesticide Residue Analysis . . . . . . . . . . . . . . . . . . . . . . . . .29
A. Sampling, Sample Handling. Storage. and Preservation .............29
B. Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 .
C. Extraction ................................................... 30
1. Purity of Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30
2. Selection of a Solvent System . . . . . . . . . . . . . . . . . . . . . . . . . . . . .30
3. Selection of a Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 .
D. Cleanup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .31
1. Liquid-Liquid Partitioning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31 .
2. Liquid-Solid Chromatography (Column Cleanup) ............32
3. Thin-Layer Chromatography (TLC) . . . . . . . . . . . . . . . . . . . . . . .34
4. Chemical Cleanup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .35
a. Acid Cleanup .................................... 35
b. Alkaline Cleanup ................................. 36
c. Base-Acid Partitioning ............................. 36
5. Sweep CO-Distillation ................................... 37
6. Gel-Permeation Chromatography (GPC) . . . . . . . . . . . . . . . . . . . 37
E. Gas-Liquid Chromatography (GLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38
l. Instrumentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38
2. GLC Column Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39
a. Column Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 .
b. Solid Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .39
c. Stationary Phase .................................. 40
d. Preparation of Column Packings ....................40
e. Packing and Conditioning of a New Column . . . . . . . . . .41
f. Carrier Gas ...................................... 42
g. Column Efficiency and Resolution ................... 42
h. Maintenance of a GLC Column ..................... 44
3. Gas Chromatographic Detectors .......................... 44
a. Electron-Capture Detector (ECD) . . . . . . . . . . . . . . . . . . .44
b. Alkali Flame Ionization Detector (AFID) and Nitrogen-
Phosphorus Detector (N-PD) . . . . . . . . . . . . . . . . . . . . . . . 46
c. Flame Photometric Detector (FPD) . . . . . . . . . . . . . . . . . . 47
d. Electrolytic Conductivity Detectors . . . . . . . . . . . . . . . . . .48
e. Microcoulometric Detector (MCD) .................. 48
111. Preparation of Standard Solutions ..................................... 48

A. Concentrated Stock Solutions ................................... 48
B. Intermediate Concentration Stock Solutions . . . . . . . . . . . . . . . . . . . . . .49
C. Workingstandards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .49
.
IV . Confirmation of Pesticide Identity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

A. Adsorption Column Elution Pattern . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
B. Thin-Layer Chromatography (TLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
C. High-pressure Liquid Chromatography (HPLC) . . . . . . . . . . . . . . . . . . . 53
D. Extraction p-Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
E. SpecificDetectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
F. Spectroscopic and Spectrometric Analysis . . . . . . . . . . . . . . . . . . . . . . . . 54
1. Nuclear Magnetic Resonance Spectroscopy (NMR) . . . . . . . . . . . 54
2. Infrared Spectroscopy (IR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3. UV Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
4. Mass Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
G. Chemical Confirmatory Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
H. Photochemical Confirmatory Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
V. Some Aspects of Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55

A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55
.
B. Purpose of Inter- and Intralaboratory Quality Control Programs . . . . .56
C. Structure of an Effective Quality Control Program . . . . . . . . . . . . . . . . . 57
D. Discussion of Some Key Aspects of an Effective Interlaboratory
Quality Control Program ...................................... 58
1. Analytical Reference Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2. Suitability of Analytical Methodology and Compatibility of
Analytical Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3. Standard Reference Materials (SRM) . . . . . . . . . . . . . . . . . . . . . . . 59
4. Sampling, Sample Handling, and Storage . . . . . . . . . . . . . . . . . . . 60
V1. Discussion of Some Key Steps in Sample Preparation for Residue Analysis ...61
A. Evaporation ................................................. 61
1. Evaporation to a Small Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
2. Evaporation by Rotary Evaporator . . . . . . . . . . . . . . . . . . . . . . . . 64
B. Replacement of One solvent for Another ......................... 64
1. For Large Volume of Solvent . . . . . . . . . . . . . . . . . . . . . . . . . . . . .65
2. For Small Volume of Solvent ............................. 65
3. For High Boiling Point Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . .65
C. Removal of Water in Water Immiscible Solvents ................... 66
D. Special Treatment of Glassware ................................. 67
E. Solventpurity ................................................ 68
F. Emulsion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .69
G. Sulfur Removal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
H. Effects of CO-Extractives....................................... 70
1. Artifacts ............................................... 71
2. Interferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
V11. Thin-Layer Chromatography (TLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
B. Adsorbents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
C. Coating of TLC Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
D. Spotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
E. Development and Visualization ................................. 74
1. Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .74
2. Developing solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .75
3. Visualization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .75
F. TLC-GLC Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .75
G. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
H. Other Forms of TLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .77
1. Reversed-Phase TLC (RP-TLC) . . . . . . . . . . . . . . . . . . . . . . . . . . .77
2. High-Performance TLC (HP-TLC) . . . . . . . . . . . . . . . . . . . . . . . .78
References ............................................................... 78
I. INTRODUCTION
Pests have long been known t o man. The Old Testament has many references to
plagues of locust, to wines "eaten by the worms", and the olive "that cast his fruit".
There are numerous forms of pests and as many attempted control measures. Formu-
lations prepared from plant extracts such as nicotine or simple inorganic salts such as
arsenic compounds were perhaps the earliest forms of pesticides recorded in ancient
documents of more than several hundreds of years ago in old countries such as China.
It was not until 1939, however, that the ability of the xenobiotic compound DDT, to
control undesirable insects was discovered. Subsequently, methoxychlor, a DDT ana-
log, was also found to be effective against a wide range of insects, although in some
instances it is less effective than DDT. Later, in 1945, the discovery of a plant growth
regulator known as 2,4-D, a phenoxyalkanoic acid, opened the door for the discovery
of a multitude of similar compounds which are used as herbicides to control undesira-
ble weeds by their selective action on broadleaf plants. Since then, chemicals for pest
control have had a dramatic rise in types, number, and quantity. Although these chem-
icals control insects, weeds, and other pests and hence increase agricultural products
and minimize diseases to humans and animals, some can also remain active in the
environment for long periods of time, and some can affect the nontarget organisms
such as fish and wildlife. The bioaccumulative tendency and nontarget side effects of
these chemicals could pose a hazard to health and to the environmental ecosystem.
Therefore, the monitoring and surveillance of these chemicals in food and in the envi-
ronment is a necessary and basic step for health protection, environmental assessment,
and pollution control. In the latter case, for example, the identification, characteriza-
tion, and measurement of the concentration of pollutants in the environment provide
not only a better understanding of the extent and effects of pollution, but also of the
effectiveness of existing and new pollution control action.
Pesticides can be defined as substances that kill or control some unwanted organisms
such as insects, fungi, undesirable plants, rodents (rats and mice), mites, or nematodes.
According to their intended targets, pesticides can be more accurately classified into
the following groups, namely, insecticides, fungicides, herbicides, rodenticides, miti-
cides, and nematocides. For example, insecticides are agents to control or kill harmful
insects affecting plants, animals, and humans; fungicides are substances that prevent,
cure, or control plant diseases caused by fungi; herbicides are substances that kill weeds
or increase or decrease plant growth or alter these states to increase their benefit to
man. These three classes of pesticides are the most widely used. Included in the general
term of pesticides could also be antibiotics, defoliants, and desiccants. An individual
chemical can have two or more functions, acting, for example, as an insecticide as well
as fungicide.
After application of these pesticides to the target area, the deposits of these pesti-
cides and their possible degradation products and metabolites integrated into edible
substances, biological systems, water, sediment, and air are called "pesticide
residues". Therefore, "pesticide residue analysis" is the qualitation and quantitation
of these residues in these matrices. Degradation products are altered pesticide mole-
cules resulting from "weathering" by chemical processes in the environment such as
hydrolysis, photochemical (sunlight) action, and oxidation. Metabolites are altered
pesticides resulting from such metabolic reactions in plants and animals as hydroxyla-
tion, demethylation, etc. However, some authors use the term "metabolites" to cover
both chemical and biochemical by-products. The most important aspect of these by-
products is that some are as toxic or more toxic or more persistent than the parent
pesticides. Therefore, they should be included in the analysis.
For pesticide applications, the above classification according to their intended tar-
gets is desirable, but for analysis, it is more convenient to classify them according to
their chemical structures even though not all the compounds with similar structures
have the same mode of action on pests. The advantage of this classification is to facil-
itate their analysis because compounds with similar structures usually respond to the
same analytical methodology.
According to their chemical structures, pesticides are classified as follows:
O.C. - This is an organochlorinated insecticide, a polychlorinated organic com-
pound such as BHC, DDT, or dieldrin.
0.p. - This is an organophosphorus insecticide, a phosphorus containing organic
compound such as parathion or fenitrothion.
herbicide acid - This is an organic compound which has a substituted phenoxyalka-
noic acid structure or its ester form such as 2,4-D or 2,4,5-T.
carbarnate - This is an ester of substituted carbamic acid which usually has a gen-
eral formula of R1R2NCOOR3(in some cases 0 is replaced by S) such as carbofuran,
carbaryl, or benomyl.
urea herbicide - This is a compound with substituted urea structure
R,R2NCONHR3, such as dinuron or linuron.
striazine - This is a herbicide with a substituted 1,3,5-triazine structure, such as
atrazine, simazine, or prometryne.
others - These other types of commonly used pesticides include uracils, chlorinated
phenols, organo-mercury and -tin compounds, as well as inorganic arsenical insecticide
and many more.
Analysis of pesticides can be grossly divided into two types, analysis of pesticides in
formulations and the residue analysis of pesticides. The former is a macro analysis to
give percentage of active ingredients present in the formulation by means of spectro-
photometric, colorimetric, and chromatographic techniques. These methods are usu-
ally not applicable to residue analysis because of the lack of sensitivity and specificity.
Pesticide residue analysis is extremely complex and tedious. It involves not only the
analysis of the parent compounds, but also their metabolites, degradation products,
or a combination of some of the above or all. Pesticide residues are often present in
minute quantities together with a large excess of interfering CO-extractives.Pesticide
residue analysis is difficult and extremely complex because of the need to isolate, ac-
curately identify, and measure such minute quantities in the presence of large amounts
of extraneous materials. However, residue analysis is needed for health and environ-
mental protection, pollution control, and so on. (See more detailed discussion in Sec-
tion V.)
Because of the complexity of residue analysis, there are two major types of analytical
uncertainties derived from them. They are (1) true identity of the contaminant and (2)
real quantity of the contaminant analyzed. The confirmation of pesticide identity will
be discussed later in this chapter and in greater detail in Chapter 3. In order to assure
Volume I 29
a
SAMPLING, SAMPLE HANDLING
PRESERVATION & STORAGE
SAMPLE PREPARATION
l i q u i d sample: l i q u i d - l i q u i d p a r t i t i o n i n g
s o l i d sample: soxhlet, blending, u l t r a s o n i c
1
a d s o r p t i o n chromatography: F l o r i s i l . s i l i c a gel. e t c
CLEANUP
chemical : a c i d , base
s u l f u r removal : mercury, a c t i v a t e d copper
TLc
GLC w i t h s e l e c t i v e l s p e c i f i c d e t e c t o r s
GCIMS
HPLC w i t h s e l e c t i v e l s p e c i f i c d e t e c t o r s
DETERMINATION
s p e c t r o p h o t o r n e t r i c methods
T
TLC
radiochemical techniques
l C chemical d e r i v a t i z a t i o n
CONFIRMATION s p e c i f i c detectors, multi-column
OF IDENTITY
6' GCfMS
I I o t h e r chromatographic techniques
+L
- ~-
i n t r a l a b o r a t o r y q u a l i t y c o n t r o l programs
CONFIRMATION i n t e r l a b o r a t o r y q u a l i t y c o n t r o l programs
OF QUANTl TY
use o f s t a n d a r d r e f e r e n c e m a t e r i a l s .
the quality of analytical data, intra- and inter-laboratory quality control programs are
required. Some fundamental aspects of quality control studies are discussed later in
this chapter.
11. GENERAL SEQUENCE FOR PESTICIDE RESIDUE ANALYSIS
A. Sampling, Sample Handling, Storage, and Preservation

Although sampling is a critical activity for generation of valid data, it is an area
where sufficient investigation and data are lacking for the best sampling procedure. It
is also an area in which the analyst usually has little control. A practical approach, as
a part of a quality assurance program, is to develop and periodically update sampling
guidelines and procedures based on in-house investigation and input from field and
laboratory personnel. This integrated approach will minimize the gap between field
and laboratory personnel.
Once the samples are properly collected in suitable containers, they should be im-
mediately transported t o the laboratory for analysis. For some analysis, preservatives
are added to the samples immediately after collection. For all analysis, proper ship-
ment procedures are needed to ensure sample integrity, that is, to ensure that the ana-
lytical data to be generated on these samples will reflect actual conditions of the sample
at the time of collection. At the present time, this is another area which requires more
investigation.
When samples arrive at the laboratory, they are not often analyzed immediately,
since the number of samples received by the laboratory often exceeds the capacity of
the laboratory at a given time. Consequently, samples are stored with or without pre-
servatives until analysis, which could be a period of several days to several months.
Proper storage conditions and preservation procedures in the laboratory are therefore
as important as those used in the field and during transit. Unfortunately, there is again
insufficient investigation regarding these aspects. In fact, it is not uncommon to find
that researchers who develop analytical methods and analysts who perform analyses
ignore these important aspects and regard analysis and sample handling (storage and
preservation) as separate entities. If valid data are to be generated, sample storage and
preservation procedures are an important and integral part of the overall analytical
scheme.
B. Sample Preparation
Before proceeding to the analysis, a sample usually requires some process to render
it into a proper form for extraction. For example, plant or animal material is usually
chopped, ground, or blended to facilitate extraction and for a large size sample, to
provide, hopefully, a homogeneous sample so that a subsample can be used for extrac-
tion. For water samples, extraction is performed as it is or with a salt added to facilitate
extraction. Sediment samples are mixed, sieved, dried, ground, or blended before ex-
traction. The sample processing procedures to be chosen depend on the sample matrix
and the parameters to be analyzed for.
C . Extraction
Since the sample generally cannot be analyzed directly for pesticide residues, extrac-
tion is required to isolate the target contaminants from the sample matrix. Extraction
in pesticide residue analysis almost always results in solution of pesticide residues and
sample CO-extractives.Extraction is preferably done as soon as the sample is collected
to avoid any possible degradation of pesticides during storage. Since this condition
usually cannot be met, all the precautions stated in the previous section should be
taken prior to extraction.
Although procedures for multiresidue analysis are known, there is no single method
or solvent system which is good for all types of sample substrates and pesticides. Below
are some of the general considerations in extraction of pesticides. Examples for indi-
vidual pesticides or pesticide classes are given in other chapters.
1. Purity of Solven ts
Solvents used in pesticide residue analyses are usually of "distilled-in-glass", "pes-
ticide residue" or "PR", and "nano" grades. They are solvents of high purity and
contain interfering impurities at or below ppb levels. Once a sample from each lot of
these solvents is tested and proved to be satisfactory, no further purification of solvents
as described in Section V1 needs to be carried out.
2. Selection of a Solvent System

The ideal solvent or mixture of solvents should be highly efficient in the recovery
of the target pesticide but selective enough not to include excessive interfering co-ex-
tractives. Often a nonpolar, polar, or a mixture of both solvents can be used and this
depends on the sample type as well as the polarity of pesticide. A choice is usually
made by running spiked recovery tests to determine which solvent system produces the
optimal recovery of the pesticide in question. However, depending on the nature of
the pesticides and/or sample matrix, spiking experiments are only regarded as rough
guidelines to the extraction efficiency of a solvent or solvent mixture and the extraction
procedure. Using the same extraction solvent, recoveries of pesticides from spiked sam-
ples can be better than those from naturally contaminated samples because the spiked
pesticides are not as well integrated into the sample matrix as the pesticide residues.
In some cases, the pesticides are tied up in the matrix as conjugates or complexes and
to free them requires more drastic procedures such as hydrolysis than straight solvent
extraction.
In all procedures the volume of the combined extract has to be reduced to a small
volume prior to determination. Thus, to ensure easy removal, solvents with lower bp
are preferred over those with higher bp, provided that they have comparable extraction
efficiency. Hence, petroleum ether or hexane is used instead of isooctane, and metha-
nol instead of propanol, etc.
Generally speaking, benzene or a mixture of hexane and acetone can be used for
the extraction of nonpolar O.C.pesticides, and more polar solvents such as dichloro-
methane or acetonitrile are used for the more polar 0.p. pesticides, carbamates, and
phenoxy herbicides.
3. Selection o f a Procedure
Again, the selection is dictated by the sample type and extraction efficiency of the
method. For water samples of 1 1 or less, extraction can be effected by shaking in a
separatory funnel or by the vortex method in a whisky bottle with a suitable solvent.
For samples of a few hundred liters in size, adsorption of pesticides on columns con-
sisting of charcoal, XAD resins, or polyurethane foams followed by desorption with
a suitable solvent can be applied. A variety of extraction methods including blending,
shaking, ultrasonic, and Soxhlet extraction have been used and shown to be effective
on spiked or fortified solid samples. However, it is generally believed that Soxhlet
extraction is the most exhaustive (i.e., highest recovery) method and thus most suitable
for weathered samples.
Once again, the procedure chosen should be tested to check recoveries for the pesti-
cides and sample type in question. It is emphasized that a procedure developed for a
particular pesticide or sample type may not be applicable to a different pesticide even
though the pesticides may be similar in structure. Spiked recovery tests will provide
some indication on the efficiency of this testing.
D. Cleanup
Cleanup is a term used in pesticide residue analysis for the isolation of the target
pesticide from interfering CO-extractives.The amount of cleanup required prior to final
determination depends on the sample type and the selectivity of both the extraction
procedure and the determinative method. For O.C. analysis using GC/ECD, sample
extracts with insufficient or no cleanup produce erratic results such as misidentification
of compounds and misinterpretation of quantities in the determination step. For 0.p.
analysis using GC/FPD, often little or no cleanup of sample extracts is required.
Again, no single universal procedure is suitable for all types of samples or pesticides.
The commonly used cleanup techniques in pesticide residue analysis are briefly dis-
cussed below.
l . Liquid-Liquid Partitioning
After the sample is extracted, the extract is usually subject to a liquid-liquid parti-
tioning step before it is cleaned up by one of the chromatographic techniques. In a
solvent system consisting of two immiscible solvents, a solute tends to partition pref-
erentially into one solvent rather than the other. The ratio of solute concentrations in
these two solvents is known as the partition coefficient. By proper choice of a solvent
system, it is possible to recover a solute or a group of similar compounds from one
solvent to another in several (usually three or less) consecutive extractions.
Solvent-solvent partitioning is a cleanup procedure used to remove significant quan-

tities of fats, waxes, lipids, and some pigment in sample extract by partitioning between
immiscible polar and nonpolar solvents. Since Jones and Riddickl in 1952 introduced
an acetonitrile-hexane partitioning for the cleanup of sample extract for Dilan (an O.C.
with a molecular structure similar to DDT), this solvent-solvent partitioning system
has become a widely used step in pesticide residue methodology.
Waxes, pigments, and lipid materials are more soluble in hexane, while the pesticides
are extracted by acetonitrile. Due to the high bp of acetonitrile, it is more convenient
to replace it with a lower boiling solvent to facilitate evaporation and minimize loss
due to prolonged evaporation of acetonitrile extract. For many pesticides, hexane is
an appropriate solvent. To decrease the solubility of pesticides in acetonitrile, water is
added before extraction with hexane.
For more polar pesticides, methylene chloride can be used instead of hexane. A
general fatty food methodology scheme described in PAM6' for O.C.S in butter, vege-
table oil, and animal fat is
l. Dissolve the fat in petroleum ether or hexane and partition several times with
acetonitrile.
2. Discard the petroleum ether or hexane layer which contains the bulk of the fat.
3. Dilute the acetonitrile with water and extract the O.C.pesticides from the aqueous
acetonitrile into petroleum ether.
We feel it is beneficial to use sodium sulfate solution instead of water in the "flooding
out" of pesticide from acetonitrile layer into the nonpolar organic layer.
Besides the use of liquid-liquid partitioning as a general cleanup procedure, it can
also provide a preliminary fractionation of a mixture of analytes into two polarity
groups. As a general example, suppose an aqueous acetonitrile extract contains residue
such as organochlorines, organophosphorus, and 2,4-D acids/esters. A hexane parti-
tioning removes most organochlorines, some organophosphorus, and 2,4-D esters res-
idues into the hexane layer, while the 2,4-D acids and some polar organophosphorus
residues or their metabolites remain in the aqueous portion. These latter compounds
may be subsequently removed from the aqueous mixture by partitioning with chloro-
form or methylene chloride in order to increase recoveries of pesticides. Saturated
sodium sulfate solution is added to the aqueous acetonitrile fraction thereby decreasing
the solubilities of pesticides in the acetonitrile/water phase, and consequently facilitat-
ing the partitioning of the pesticides into the organic phase. It should be noted that
since the CO-extractivesmay or may not alter the partition coefficient of a pesticide in
a certain solvent system, spiked recovery experiments involving the sample matrix in
question should be carried out to verify that the recovery is adequate.
Instead of acetonitrile, dimethyl formamide (DMF) and dimethyl sulfoxide (DMSO)
have been used with hexane or petroleum ether for liquid-liquid partitioning cleanup.
These solvent systems are compared briefly by Thornburg2 for the extraction of 0.c.s
in butter oil. In general, there is a greater tendency for DMF or DMSO than acetoni-
trile to form emulsion when partitioning with hexane, but in some cases, recoveries
are better under the same extraction conditions. The application of liquid-liquid par-
titioning t o cleanup sample extracts for carbamates, herbicides, and 0.p.s will be dis-
cussed in the respective chapters.
2. Liquid-Solid Chromatography (Column Cleanup)

Undoubtedly, the primary objective of using an adsorption cleanup column in pes-
ticide residue analysis is to remove pigments, waxes, polar impurities, and small
amounts of fats and liquids from one sample extract. By means of graded elution, i.e.,
Volume 1 33
elution of the column with a less polar solvent first, followed by elution with one or
several more polar solvents, in the order of increasing polarity, fractionation of pesti-
cides can be effected (see Table 7 on eluotropic series of solvent in Section VII). The
grouping of pesticides into several portions facilitates, in a multi-residue analysis, the
identification and determination of the pesticides by GLC. Also, the elution pattern
in the adsorption column provides useful information on the confirmation of pesticide
identities.
Chromatographic columns can be packed dry or as a slurry in an appropriate sol-
vent. The column packing must be uniform and free from voids. Channeling of solvent
will occur in the presence of the latter, producing poor separations. It is often useful
to place l-in. layers of anhydrous sodium sulfate at the bottom as well as the top of
the adsorbent. This ensures that the column eluate is dry and thus compatible with
most o f the GLC systems (such as GLC/ECD) which are used in subsequent determi-
nation.
The adsorbents most extensively used in residue analysis are alumina, silica gel, and
FlorisilC?. A comprehensive review on these adsorbents and their application in pesti-
cide residue analysis has been given by M ~ r l e yRepresentative
.~ examples for individual
pesticides are given in the corresponding sections in the following chapters.
Alumina - Activated alumina is prepared by the partial dehydration of aluminum
hydroxide at about 400°C for 4 hr. The alumina thus prepared is basic and very hydro-
scopic. For some compounds, aldol condensation, dehydration, dehydrochlorination,
etc. will take place during column chromatography on this basic column. In such cases,
neutral or acidic alumina would be required for the separation of alkali-labile pesti-
cides. Neutral alumina is obtained by washing the basic alumina with distilled water
until the extract is neutral. When acidic alumina is required, the adsorbent can be
obtained by suspending alumina in an acid until the extract has a pH 4 to 6. Standard-
ization of alumina can be carried out by the technique of Brockmann and Schodder4
using azo dyes. Partial deactivation of Brockmann grade I or Woelm alumina is
achieved by the addition of up to 15% of distilled water by weight (Table l), thorough
mixing on a rotary mixer for 2 hr and equilibration of content overnight before use.
Reactivation of deactivated alumina can be done at 150 to 200°C.
Silica gel - Silica gel, also known as silica or silicic acid, has the general formula
SiO, X H,O and is made up of porous three-dimensional siloxane structures with polar
surface silanol, Si-0-H groups. Activation of silica gel by heating to about 200°C,
components adsorbed physically on the bound hydroxyl groups are released and on
further heating to about 400°C, the free hydroxyl groups may liberate a molecule of
water t o form a less active adsorption site consisting of an oxygen bridge between two
silicon atoms. In agreement with this, an increase in silica gel activity was observed
on activation by heating to 200°C, while activations beyond this temperature led to a
decrease in adsorption of substances that can form hydrogen bonds. In a study on the
activation and standardization of high purity grades 950 and 923 silica gel, Kadoums
reported that adsorbents activated at 130" or 300°C for a 2-hr period were both satis-
factory in terms of separation of pesticides from biological materials and pesticide
recovery. No gain in activity could be obtained by prolonged heating and the silica gel
thus prepared showed no change in activity for as long as 60 days when it was placed
in a tightly closed container. Deactivation of silica gel is accomplished by the addition
of various percentages of water to the activated adsorbent. Successful cleanup proce-
dures using silica gel have been applied to residue analysis of 0.c.s and certain 0.p.s.
FlorisilB - FlorisilB is a synthetic magnesium silicate manufactured by Floridin
Company. Normal activation at 650°C and storage at 130°C just prior to use are nec-
essary to keep FlorisilB in the most active form. The adsorbent is most widely used
for column cleanup and separation of chlorinated pesticides and detailed discussions
on the characteristics and applications of FlorisilB are given in Volume 11, Chapter
l . Besides O.C.pesticides, FlorisilO is also used in the cleanup of many pesticide resi-
dues including carbamates, triazines, phenoxy acid esters, and certain but not all 0.p.s.
3. Thin-Layer Chromatography (TLC)

TLC is primarily a type of adsorption chromatography where the adsorbent is coated
as a thin layer onto a rigid support such as glass plates, aluminum sheets, and celluloid
plates. Like all techniques of chromatography it involves two phases. The mobile phase
(developing solvent or mixture of solvents) is in equilibrium with the stationary phases
(adsorbents). Commonly used adsorbents are finely graded alumina or silica gel, usu-
ally mixed with a binding agent such as starch or gypsum to add greater mechanical
stability to the adsorbent layer coating on the plates.
Briefly, concentrated sample extract or standard solution is placed at a short distance
from one end of the TLC plate which is then placed in a closed chamber (developing
tank) containing a solvent or a mixture of solvents, known as a developer. By capillary
action passing through the layer, different compounds travel at different rates on the
plate and hence effect separation. The plate is then taken out of the developing tank.
After evaporation of the developing solvent(s), the compounds are located under UV
light or by a spraying reagent.
A compound is characterized by the distance it travels from the point of application
(bottom of the plate) in relation to the solvent front (top portion of the plate), R,
values are used to characterize a compound under specified TLC conditions such as
developing solvent, temperature, and type of adsorbent. R, value is defined as the
distance traveled by a compound divided by the distance traveled by the solvent front.
The distance traveled by the spot is determined by measuring from the point of appli-
cation to the center of the spot at the point where it finally comes to rest. Sometimes
heating the sprayed plate in the oven is necessary in order to visualize the compounds
which show up as spots on a TLC plate. The solvent front is determined by immedi-
ately marking the distance the solvent has traveled after the TLC plate has been re-
moved from the developing chamber and before the solvent has evaporated.
A chromogenic spray reagent is a soltuion of a single chemical or a mixture of chem-
icals which reacts with the compound on the TLC plate to form a color spot so that
visual location and identification of the compound is possible. Unlike the use of the
acid charring technique which is a totally destructive method, chromogenic sprays are
often semidestructive in their application; in other words, the original molecules react
to form color derivatives.
In pesticide residue analysis, TLC is more commonly used as a separation or cleanup
technique than for quantitative analysis. This is because TLC is only semiquantitative
and, in general, less sensitive than GLC technique.
T o apply TLC as a cleanup technique for sample extract, the standard and extract
are run side by side. Better still, the extract is run between two identical standards.
After development, the plate is covered with a cardboard, so that only the area repre-
senting the standards is exposed to the chromogenic spray reagent or UV irradiation.
After locating the area of the standards, the corresponding area on the sample portion
of the plate is removed by a microzone collector which scrapes and removes the adsor-
bent by vacuum suction in the appropriate area. A zone collector operates on the same
principle as a vacuum cleaner and the scraped adsorbent is deposited in a removable
extraction cylinder. A water aspirator is usually the suitable vacuum source. The ad-
sorbent can then be extracted with an appropriate organic solvent, and after proper
concentration, the extract can be examined by gas chromatography.
Cleanup of sample extract by TLC is an alternative means of reducing background
interferences for gas-liquid chromatographic analysis. However, this technique is not
as widely used as column chromatographic technique for cleanup and separation of
Volume I 35
Table 1
ACTIVITY SCALE
O F ALUMINA AS A
PERCENTAGE O F
WATER CONTENT
Brockmann Percentage
grade water
I (Woelm) 0
II 3
111 6
1v 10
V 15
Note: Alumina has been ap-

plied in the residue
analysis of various
samples containing
o.c.s, o.p.s, triazines,
and other pesticides.
pesticides in a sample extract, mainly because of the limited sample loading capacity
and resolution power. Two-dimension TLC increases the resolution power consider-
ably but it is quite time consuming (see Section VII).
For dirty sample extracts, after the usual column cleanup and fractionation of pes-
ticide residues, further cleanup by TLC is often advantageous. Used in conjunction
with GLC, TLC is also useful for providing additional substantiation to residue iden-
tity.
A more detailed discussion on the basic technique and principle will be presented in
Section VII.
4. Chemical Cleanup
Cleanup by chemical reactions of the sample extract offers a useful alternative to
cleanup by column or thin-layer chromatography as discussed above. However,
cleanup by chemical means is a more specific approach and does not have the wide
scope of application of the chromatographic cleanup procedures.
Generally, chemical cleanup can be classified into two main groups, acid and alka-
line cleanup procedures. In both approaches, the sample or sample extract is treated
with either a strong mineral acid or alkali to destroy or remove interfering materials.
Obviously, the analytes in question must be inert to this acidic or alkalinic treatment.
Since most pesticide residues are labile under these conditions, the scope of application
by the chemical cleanup procedures is limited mainly to some stable o.c.s, acid herbi-
cides, and phenols.
a. Acid Cleanup
A notable example of the application of acid cleanup is on the elimination of inter-
ferences in the determination of toxaphene re~idues.~.'Toxaphene is a complex mixture
of chlorinated camphene. The GLC chromatogram consists of many peaks overlapping
several O.C. peaks, particularly in the DDT-DDD region. Since the ECD response to
DDT is several times greater than to an equal amount of toxaphene, the degree of
interference caused by DDT in a GLC analysis of toxaphene is disproportionate to the
amount of DDT present. Treatment of sample extract with cold sulfuric-fuming nitric
acids6 removes DDT interference by converting it to compounds which do not register
on the GLC. Toxaphene is hardly affected and responds in the usual manner. How-
ever, chlordane is not removed by the acid treatment and interferes in the analysis.
Similar acid treatment has been used t o eliminate interference of DDT, DDE, DDD,
and sample CO-extractivesfor PCB8-'O and PCTLO analyses. PCBs (polychlorinated bi-
phenyls) and PCTs (polychlorinated terphenyls) are industrial compounds of recent
environmental significance. They are very inert nonpolar mixtures and are not affected
by the acid treatment under the condition used. Similarly, using a more drastic acid
treatment (fuming sulfuric acid and nitric acid at elevated temperature), PCBsL1~" can
be nitrated, but the much more stable mirex, an o.c., remains intact. The highly polar
nitrated PCBs can then be easily separated from the nonpolar mirex. Thus the inter-
ference of PCBs on mirex analysis is eliminated.
b. Alkaline Cleanup
Under various conditions alkalis such as KOH or NaOH have been used t o remove
interfering CO-extractiveso r specific compounds. For high lipid or fat sample extracts
such as fish and sea gull tissues, treatment of sample extracts has been used to remove
these interfering materials by converting them to water soluble "soap" which is easily
removed from the water-insoluble O.C. pesticides and PCBs. However, several o.c.s,
notably the DDT group, are affected t o greater or less degree by alkaline treatment
(see Chapter 3 for detailed discussion), and of course the generally more labile 0.p.s
and carbamates will be more seriously affected. Therefore, like the acid cleanup, the
alkaline treatment is applicable only to certain specific situations. A noteworthy ex-
ample for its application is the removal of interference of DDT and its analog^'^.'^ in
the analysis of PCBs. In this approach, DDT and analogs (DDD, methoxychlor, etc.)
are dehydrochlorinated with an alkalineL3(ethanolic KOH) or DBUL4(1,5-diazobicy-
clo-r5.4.01 undec-5-ene) to DDE which is subsequently oxidized by CrO, to the polar
dichlorobenzophenone. The intact nonpolar PCBs can be easily separated by column
chromatography from the more polar 4,4'-dichlorobenzophenoneby applying the ion-
pair two-phase reagent technique. DDT and analogs can be dehydrochlorinated and
oxidized in one step15 using tetrabutylammonium permanaganate containing sodium
periodate and sodium hydroxide. Again, the final product, dichlorobenzophenone can
be easily separated from the intact PCBs thus eliminating the interfering peaks from
DDT and analogs.
c. Base-Acid Partitioning
The classical technique of using acid t o extract basic compounds or base t o extract
acidic compounds is applied to pesticide residue analysis for both extraction and
cleanup purposes. Since there are many more acidic pesticides (phenols, phenoxy, and
carboxylic acid herbicides) than basic pesticides, base-acid partitioning is more com-
monly encountered. In brief, a sample is usually acidified to ensure that the acidic
compounds are in the acid form. The sample is then extracted by an appropriate sol-
vent such as chloroform, methylene chloride, or ethyl acetate. The organic extract will
contain both neutral (o.c.s,16 o.p.s, carbamates") and acidic pesticides. The acidic
pesticides are subsequently removed and separated from the neutral compounds by
forming water-soluble salts with an alkaline solution (such as a solution of sodium
hydroxide, carbonate, or bicarbonate). Acidification of the alkaline extracts will regen-
erate the acids which are then extracted into an organic solvent. The organic extract
can then be processed for analysis. This base-acid partitioning eliminates any possible
interference from neutral compounds and neutral sample CO-extractives. It is a partic-
ularly popular step in analytical methods for the analysis of acid herbicides.
In summary, the application of chemical reactions for cleanup purposes does not
have a general scope of application. This approach is suitable only for a specific pur-
pose and a specific type of compounds. In modern surveillance and monitoring activi-
ties, there is a growing need for multi-class and multi-residue analytical methods and
nondestructive techniques such as column chromatography for general application.
Chemical reactions which assist in the analysis of a few compounds at the sacrifice of
others are usually regarded as supplementary techniques rather than as the basic
scheme for cleanup.
5. Sweep CO-Distillation
In the sweep CO-distillationtechnique a sample extract in a suitable solvent, such as
ethyl acetate, is injected into a heated glass tube packed with glass wool; the injection
is followed by several repeated injections of ethyl acetate at several-minute intervals.
Nitrogen gas, acting as carrier, sweeps through a cooling bath of ice and water into a
short Anakrom scrubber tube and finally t o a concentration collection tube. The basic
principle of this technique is the assumption that the sample CO-extractivesare depos-
ited onto the glass wool, whereas the pesticides are volatilized and collected in a con-
centration tube. This technique was primarily designed for the cleanup of sample ex-
tracts for 0.p. analysis. Since 0.p.s are quite volatile in relation to most sample co-
extractives such as waxes, lipids, and pigments, this is an attractive technique for o . ~ .
analysis. In practice, this cleanup technique was found to be suitable for P-specific
detectors and not vigorous enough for ECD. However, as discussed in Volume 11,
Chapter 2 on o.p.s, little or no cleanup is needed for the analysis of 0.p.s in many
types of plant and food samples if FPD is used. If cleanup is required, liquid-liquid
partitioning is quite often sufficient.
Sweep CO-distillationhas been extended to several 0.c.s but not to other classes o f
pesticides such as ureas, a d d herbicides, and carbamates, which are either nonvolatile
or easily decomposed thermally.
6. Gel-Permeation Chromatography (GPC)

In the removal of fat and lipids from sample extracts, we have discussed (1) adsorp-
tion column,or thin-layer chromatography using adsorbents such as FlorisilB, silicic
acid, and alumina, (2) liquid-liquid partitioning using solvents such as acetonitrile and
hexane, and (3) sweep CO-distillation(volatilization). Low-temperature precipitation of
lipids such as the procedure developed by McLeod and wale^'^ is another technique.
The recent development of an automated gel-permeation chromatographic system19
offers another alternative cleanup procedure.
Unlike separation in adsorption chromatography (TLC or column), which is based
on polarity of the analytes, separation in GPC is based on the different abilities of the
various sample molecules t o enter pores in the gels. Very large molecules which never
enter the pores will pass through the gel layer the fastest. The smallest molecules will
enter the pores in the absorbent and spend the longest time in it before passing through
the column layer. The time spent by molecules in the pores of the gels is inversely
proportional t o their size. Thus molecules are eluted in order of decreasing molecular
size. The adsorbents used are some form of gel and the technique of the separation is
some kind of permeation or filtration; hence, it is called gel-permeation or filtration
chromatography.
The molecular weights of most pesticides are between 200 and 400, while those of
most lipids are between 600 and 1500. Also, molecular volumes of chlorinated pesti-
cides of high molecular weights are smaller than the lipid moleculars of similar molec-
ular weight.
A good example is mirex, which has a high molecular weight (545.59) yet a compact
molecular size because of the cage-structure. Thus G P C can separate lipids or other
high-molecular-weight CO-extractivesfrom most pesticides.
The efficiency of GPC using the automated system developed by Stalling et al.19,20
has been compared with the acetonitrile hexane (or petroleumn ether) partitioning and
FlorisilB cleanup procedure of PAM. Griffith and Craunz' stated that for the 30 pes-
ticides investigated (o.p.s, o.c.s, and a few other pesticides), automated GPC using
cyclohexane as eluting solvent generally gives better recoveries. However, the recover-
ies of several pesticides are considerably lower by the G P C procedure (see Table 1 of
Reference 21). Later Johnsonzz modified the elution solvent by using toluene/ethyl
acetate, which much improved the recoveries of pesticides from GPC. The recoveries
are summarized in an anonymous reportz3 by Analytical Biochemistry Laboratories,
Inc.
It may be noted that G P C is only a cleanup procedure to separate lipids or waxes
from pesticides. The lipids, being higher in molecular weight, are eluted first. GPC
does not fractionate pesticides into groups for subsequent GLC analysis. The general
practice is to use G P C to remove lipids, and the pesticides are further fractionated by
FlorisilO or silicic acid column chromatography before GLC examination. For non-
fatty samples such as water and sediment, there is usually no advantage in using GPC
as a cleanup procedure.
E. Gas-Liquid Chromatography (GLC)

l . Instrumentation
The gas chromatograph is the most important and widely used instrument for rou-
tine pesticide residue analysis. Apart from the column and detector, modern gas chro-
matographs usually contain the following basic units or modules:
1. Flow controller. It provides a constant flow rate for carrier gas through the col-
umn during operation. Erratic flow rate would cause unnecessary misinterpreta-
tion o f the quality and quantity of the anlayte. Most gas chromatographs are
equipped with rotameters but they do not give an accurate measure of flow rate
because they are installed ahead of columns. Flow rates can be checked by using
a soap bubble flow-meter attached to the detector exit.
2. Injection port. There are two types of injection ports designed to accommodate
either on-column or off-column injection. In the former case, the sample is di-
rectly injected onto the glass wool plug of the column inlet. In the case of off-
column injection, the sample is injected into a metal or glass insert installed in
the injection port, and then flash vaporized and swept into the column by the
carrier gas. For frequent injection of uncleaned samples or biological extracts,
the off-column injection with removable glass inserts is the method of choice. In
this case, the insert will trap most dirt from the sample, thus preventing contam-
ination of the column. Those inserts should be removed for cleaning on a regular
basis. Dirty inserts are known to greatly reduce column efficiency and sensitivity
as well as to cause decomposition of pesticides. For pesticide analysis, metal in-
serts which can cause catalytic decomposition of labile pesticides such as DDT
and endrin should not be used. However, when an on-column derivatization tech-
nique is needed for the analysis of pesticide, an on-column injection port with a
separate heater control must be used.
3. Temperature controls and selection. Temperature controls are usually provided
for the regulation of temperatures of the injection port, detector, and column.
On older models, those controls are usually of analog type (continuously varia-
ble) and they have the disadvantage that repeating a temperature setting accu-
rately is difficult. Newer models have digital temperature controls and thus reset-
ting to a previously determined temperature is always easy and reproducible.
Temperatures at various selected parts of a gas chromatograph are monitored by
thermocouples and are indicated by either a pyrometer or a digital readout.
Volume I 39
Many gas chromatographs are equipped with a temperature programmer, which en-
ables the user to have a temperature programmed analysis instead of just isothermal
operation. In the analysis of a complex mixture containing compounds of very differ-
ent volatility, temperature programming provides a much improved resolution of the
compounds at a shorter analysis time. However, temperature programming is not com-
monly used in conjunction with an electron-capture detector operating at high sensitiv-
ities (typical of most pesticide residue analysis) because of different bleeding rates of
most liquid phases at different temperatures, which then cause a drifting baseline.
Low-bleed DexsilB columns are exceptions in this case.
The importance of a constant oven temperature has been emphasized by many au-
thors. A properly operating oven should maintain the column temperature to a devia-
tion of r O . l "C. Erratic column temperature would cause changes in relative retention
times and thus misidentification.
The injection port is usually set at a temperature 30 to 50°C above column temper-
ature to prevent condensation of the sample in the injection port. Too high an injector
temperature would cause decomposition of heat-labile compounds, excessive septum
bleed, and reduced septum life. The detector temperature should also be at least 30 to
50°C higher than column temperature to prevent condensation of samples and column
bleed in the detector. Most often, the selection of a detector temperature is dictated
by the design of the detector. An improperly chosen detector temperature gives noisy
response and reduced sensitivity. Column temperatures are chosen to give the required
resolution of the compounds of interest at a minimum time, keeping in mind that the
minimum and maximum operating temperatures of the liquid phase are not exceeded
(see later discussion).
2. G L C Column Technology
a. Column Material
Besides borosilicate glass, various kinds of tubing materials are commercially avail-
able and used. These include copper, aluminum, stainless steel, nickel, TeflonB, and
TeflonB-lined stainless steel. Although these materials may prove satisfactory in many
other applications, the best column material for pesticide residue analysis is glass be-
cause of its inertness. Another advantage of glass column is that the condition of the
column packing can be easily inspected. Metallic columns, especially those made of
copper and stainless steel, cause on-column catalytic degradation of pesticides. For
example, as much as 25% degradation of p,p'-DDT results from a stainless steel col-
u m a Z 4A brand new glass column should be cleaned with chromic acid, then washed
with water and distilled water. After it is dried, the column should be silanized with a
10% dimethyldichlorosilane (DMCS) solution in toluene in order to remove surface
silanol groups which form hydrogen-bonds with polar compounds and cause tailing.
It is then washed with methanol and dried in an oven before it is packed. Columns of
1.2 to 3.6 m in length and 2 to 4 mm in internal diameter are commonly used in GLC,
with the 1.8 m X 2 o r 4 mm internal diameter being most popular.
b. Solid Support
Solid supports used in gas-liquid chromatography must meet the following criteria:
1. Be inert to prevent catalytic decomposition of labile compounds, adsorption, and

tailing of peaks
2. Be porous or permeable to permit reasonable carrier gas flow rates without ex-
cessive pressure drops
3. Have small and even particle size to improve column efficiency (see later discus-
sion)
Flux-calcined diatomaceous earth materials such as Anakrom, Chromosorb W HP,

Gas Chrom Q, SupelcoportC3, and others are often used as solid supports in pesticide
analysis. Supports are acid-washed to remove traces of metallic impurities which cause
catalytic degradation and then silane-treated to eliminate surface silanol groups which
cause peak tailing. Supports of mesh sizes of 60 to 80, 80 to 100 and 100 to 120 are
being used; the last one is preferred because of its higher efficiency (see later discus-
sion).
c. Stationary Phase
The stationary phase is a material which remains stationary in a GC column while
the injected solutes partition between it and the mobile (or gas) phase. In GLC, the
stationary phase is a liquid at the operating temperature and hence it is also called the
liquid phase. It is coated on a support in a packed column, or onto the tube wall in
the case of an open tubular capillary column.
There are over 200 stationary phases available commercially, although some of them
have found more applications than others. Each phase is characterized by its lower
and upper temperature limits, as well as by its polarity, which is defined by the Mc-
Reynolds' constants.25The temperature limits define the lowest and highest operating
temperatures one can use in the GC analysis. Below the lowest limit, the stationary
phase is either a solid or very viscous liquid and peak broadening will occur. Operation
beyond the upper limit results in excess column bleed which increases the noise, short-
ens the column life, and also exposes the active sites of the solid support to cause peak
broading and adsorption of analyte. The McReynolds' constants are measurements of
relative retention indexes and they serve as an indication for phase selectivity and po-
larity. Generally speaking, phases with similar constants would have similar separation
characteristics, although some exceptions may occur.
The choice of stationary phase is largely governed by the resolution and temperature
requirements of the sample and is mainly accomplished by trial and error. In general
terms, nonpolar compounds are better resolved on low polarity stationary phases and
vice versa. Among phases of similar polarity, the one with better thermal stability (less
column bleeding) is the choice. The percentage loading of stationary phases on solid
supports was as high as 40% in the past but a total loading of 3 to 10% liquid phase
is more commonly used at present. Properties of a few most frequently used liquid
phases in pesticide analyses are given in Table 2.
d . Preparation of Column Packings

Several methods are available for the making of column p a c k i n g ~ These
. ~ ~ include
the so-called pan-coating, filtration coating, and fluidization techniques as well as ro-
tary vacuum technique. The rotary evaporator technique which is recommended by
many workers is briefly described here.
1. Determine the weights of stationary phase and solid support as required by the
percentage loading and the amount of packing.
2. Dissolve stationary phase in a suitable solvent (determined by the stationary
phase) and combine with another liquid phase solution into a round bottom flask
if a mixed-phase packing is required.
3. After complete dissolution, slowly pour the solid support into the solution of
stationary phase so that the particles are evenly wetted.
4. Attach flask to a rotary evaporation with just enough suction to hold flask, and
rotate slowly for evaporation at room temperature for 10 min.
5. Bring the water bath temperature to 40 to 50°C with gradual increase in vacuum.
6. When the solvent is nearly dry, apply full vacuum to remove all solvent.
Table 2
PROPERTIES O F SOME COMMONLY USED STATIONARY
PHASES IN PESTICIDES RESIDUE ANALYSIS
Maximum
temperature
Phase Structure ("c) Polarity
SE-30 Methyl silicone, gum low

ov-l Methyl silicone, gum low
ov-101 Methyl silicone, fluid low
OC-200 Methyl silicone, fluid low
SP-2100 Methyl silicone, fluid low
DexsilF 300 Carborane/methyl silicone low
Dexsil@ 400 Carborane/methyl phenyl silicone medium
OV-17 Methyl phenyl silicone medium
SP-2250 Methyl phenyl silicone medium
ov-210 Trifluoropropyl silicone medium
QF- l Trifluoropropyl silicone medium
SP-2401 Trifluoropropyl silicone medium
CarbowaxC? 20M Polyester high
DEGS Polyester high
Silar IOC Phenyl cyanoalkyl silicone high
7. When the packing is visibly dry, release vacuum slowly and transfer packing to
a n oven.
8. Bake packing a t 110°C for at least 2 hr to complete drying.
This method is particularly useful for the preparation of mixed-phase packings when
a common solvent for both stationary phases is difficult to find. Also, it is easier to
operate when the solution of stationary phase becomes too viscous to filter as required
by the filtration technique.
e. Packing and Conditioning o f a New Column

A column can be packed by either mechanical vibration or vacuum suction. Before
it is packed, the internal wall of the column should be cleaned, silane-treated, and
dried as described above. For U-shaped columns, either of the above methods can be
used for packing a column. However, vacuum is about the only way to pack a coiled
column.
Mechanical vibration - Pour a few inches of column packing into each end of the
column through a glass funnel with a short piece of TeflonB tubing attaching to the
end. Tap the column gently with a pencil t o obtain a firm and uniform packing. Repeat
this step until the column is filled to just below the SwagelokO nut when column is
installed in the GC oven. Plug both ends with 1 cm of silanized glass wool.
Vacuum suction - Plug the detector end of the column with 1 cm of silanized glass
wool and connect the exit end to an aspirator by means of a rubber or TeflonO tubing.
Apply slight vacuum to the column and suck the packing material in increments into
the column by dipping the column inlet end into the packing material. Some gentle
tapping is often required to settle the packing. After the column is filled, plug the inlet
end with 1 cm of silanized glass wool.
In order to obtain high efficiency and reproducibility, the following points should
be kept in mind during the packing of a column:
1. If the column is intended to be a certain length, it should be packed to that length

but not less.
2. For reproducible results, columns packed with the same batch of packing mate-
rial should have very similar packing density.
3. During packing of a column, strong suction and vigorous mechnaical vibration
should be avoided. Otherwise, the coated packing may break and adsorption/
degradation of sample will occur. Also, strong suction and vigorous vibration
will produce a t o o densely packed column which has low permeability and thus
lower efficiency.
4. Glass wool plugs a t both ends of the column should only be tight enough to
prevent dislodging of packing material. Overly tight glass wool plugs create ex-
cessive back pressure when carrier gas is applied.
Before a newly packed column can be used routinely for trace analysis, it should be
conditioned in a n oven a t a temperature of 30 to 50°C o r so higher than the intended
operating temperature with a small carrier gas flow rate for a t least 24 to 72 hr. For
some nonpolar phases such as OV-l a n d SE-30, the so-called "no flow conditioning"
can be applied prior t o the above heat curing. In this case, the column is heated to
25°C below its upper temperature limit with n o carrier flow for 1 hr and then rapidly
cooled down to room temperature before it is further conditioned with flow. In all
cases during conditioning, the detector end of the column should be disconnected to
avoid contamination of the electron-capture detector.
Usually the column efficiency and response gradually improve as the column is being
used. In some cases, "priming" of a new column by several injections of a more con-
centrated (e.g., a 100 times o r more) standard solution also enhances the response.
However, with more stable liquid phases and high performance solid supports now
available, this "priming" is usually not necessary. A well-maintained column should
have a usable life of a t least a few months.
f . Carrier Gas
It has been a common practice that nitrogen and 5 to 10% methane in argon are
used as carrier gases with electron-capture detectors to avoid metastable effects. (See
later discussion o n electron-capture detector.) In all cases, the gas must be of high
purity (better than 99.995%) a n d free of traces of oxygen and moisture. The presence
of oxygen a n d water as impurities in the carrier gas will oxidize and hydrolyze, respec-
tively, the stationary phase a t elevated temperatures. These reactions will shorten col-
umn life a n d reduce column efficiency. The occurrence of oxygen in carrier gas also
reduces the sensitivity for electron-capture detection by reducing the standing current.
As little as 10 ppm of oxygen in a nitrogen carrier was reported to reduce the standing
current by 50%." Moisture in the carrier gas can be removed by passing the gas
through a filter-drier element such as a molecular sieve cartridge. Oxygen impurity can
be eliminated by using a commercial oxygen trap. Further comments on carrier purity
in gas chromatography have been discussed by Perretta."
g. Column Efficiencyand Resolution

T h e efficiency of a GC column is defined by the total number of theoretical plates,
N, of the column calculated with respect to a certain chromatographic peak by the
following equation:
Volume 1 43
where L is the length of column, H (or HETP) the height equivalent to a theoretical
plate, t, the retention time of the peak, and W the width of peak at the base. This
equation is also expressed as:
where W,,, is the width of peak at half height. The larger the N, or the smaller the H,
the narrower is the peak. The theoretical plate height, H, can be expressed in terms of
column parameters given in the famous van Deemter equation. In an abbreviated
form, the latter can be written as:
in which A is known as the Eddy diffusion term, B the longitudinal diffusion term,
C, the resistance to mass transfer of solute in the liquid phase, C, the resistance to
mass transfer of solute in the mobile phase, and G is the average linear velocity of the
carrier gas. A detailed discussion of this equation is beyond the scope of this chapter
and the reader is referred to some recent review^.^^,^^ In general terms, one can consider
the following factors in the attempt to improve column efficiency and resolution.
1. Support particle size. Higher column efficiency is obtained with the more closely
sized support, e.g., 70 to 80 mesh instead of 60 to 80 mesh. Also column effi-
ciency is improved as particle size of the support is decreased. Since in the van
Deemter equation both A and C, are directly proportional to d,, the support
particle diameter, HETP decreases or column efficiency increases when d, is
smaller. Also, the smaller the support particle, the better the column can be
packed. However, particle size cannot be reduced indefinitely for the same pack-
ing density, and the column permeability is proportional to dp2.A good compro-
mise would be use of 100 to 120 mesh size which gives good efficiency without
excessive back pressure. Permeable columns permit more rapid analysis; for the
same outlet velocity the pressure gradient is smaller and the average gas flow
velocity u is higher.
2. Column internal diameter. It has been shown that packed columns with smaller
internal diameter are more efficient than those with larger internal diameter. It
is for this reason that the 2-mm internal diameter columns are preferred to the
4-mm columns. A result of smaller internal diameter is the requirement of a
smaller flow rate, which beneficially affects the column efficiency as well as the
detector response.
3. Loading of stationary phase. It can be shown that a lower loading of stationary
phase reduces the C, term of the van Deemter equation and thus increases column
efficiency. A smaller amount of liquid phase also reduces retention time and gives
faster analysis. On the other hand, too low a loading increases the potential prob-
lem of exposing uncoated active sites on the solid support and also results in
lower capacity of the column, which limits the sample sizes to be injected. A
small sample size makes the error due to adsorption of solute on column more
critical. Therefore, in choosing or designing the loading of a column a compro-
mise is made between these opposing factors. In general a total loading of up to
about 10% liquid phase is a good compromise for pesticide analysis.
4. Column length. The resolution of peaks is proportional to the square root of
column length. A longer column also provides higher total plate number. At a
constant inlet pressure, an increase in length will result in an increase of retention
time. If the van Deemter plot (i.e., H vs. ii curve) is relatively flat, one can in-
crease the column length and carrier velocity. This will improve resolution while
keeping the analysis time down. However, if the increase in H is as large as the
-
u increase, then only the column length should be changed to improve resolution.
5. Operating conditions. In many cases, lower flow rates and lower column temper-
ature improve column efficiency as well as resolution. This is sepecially effective
when working with columns o f low loading which require smaller flow rates and
lower temperatures for adequate elution time.
h. Maintenance o f a GLC Column

In order to ensure stability and performance of a GLC column, the following prac-
tices and precautions should be exercised:
1. Impurities such as water, air, or oxygen in the carrier gas should be eliminated
as described earlier. The presence of such impurities would deteriorate the sta-
tionary phase and hence shorten the useful life of a column.
2. Periodic replacement of septum will assure leak-free performance.
3. If injection of biological samples and samples with little or no cleanup is fre-
quent, the injection liner, glass wool plug, or the first few inches of packing at
the injector end should be replaced regularly. Dirty injection liner or glass wool
would cause much reduced efficiency and decomposition of labile compounds
such as DDT.
4. If a column is left in an oven for continuous operation, periodic checking for
loose SwagelokB nuts is necessary. A tight and leak-free column installed 1 or 2
weeks ago may become loose after use. Leaky column connections give erratic
and noisy baseline, changing retention times and detector response.
5. Silylation with Silyl8 is not desirable for flame photometric detector (see Volume
11, Chapter 2) but is useful for O.C. analysis. It removes surface silanol groups
which cause on-column degradation and hence much reduced response of endrin.
CarbowaxB treatment immediately after column conditioning provides im-
proved response for organophosphorus pesticides. Repeated treatments cause a
change in relative retention times and are therefore not recommended.
6. Columns removed from the GC oven should be capped at both ends to avoid
contamination. They should be reconditioned for a few hours upon reuse.
3. Gas Chromatographic Detectors

The two governing requirements for GLC detectors in pesticide residue work are
sensitivity and selectivity. Ideally, the detector should be sensitive to trace amounts
(e.g., ng t o pg) of the pesticides in question but selective enough so that sample CO-
extractives would not interfere with the qualitation and quantitation. Specific detectors
not only improve the quality of experimental results but also minimize sample cleanup.
The theory, development, and operating characteristics of GLC detectors have recently
been r e ~ i e w e d . ~A' .brief
~ ~ summary of the most popular detectors used in the detection
and quantitation of pesticide residues is given below.
a. Electron-Capture Detector (ECD)

Inside the electron-capture detector, a radioactive source expels electrons towards
the anode. Collection of the electrons creates a current called standing current, which
is of the order of lO-' amp. If a sample which can absorb electrons is eluted through
the detector, the standing current will decrease. The decrease in standing current results
in a peak on the recorder by way of amplification in the electrometer. Because of the
nature of the design, the ECD has extremely high sensitivity towards halogenated com-
Volume I 45
Table 3
RELATIVE RESPONSE FACTORS OF
ECD
Response Functional group
One Hydrocarbons (e.g., hexane, benzene)

To lOX Ethers, esters
To lOOX Aliphatic alcohols, ketones, amines,
monochlorides, and monofluorides
To lOOOX Dichlorides, difluorides, and
monobromides
To 104X Trichlorides, anhydride
To 105X Monoiodides, dibromides, and
nitrocompounds
To 106X Diiodides, tribromides, polychlorides,
and polyfluorides
From Hewlett-Packard Operating Manual for Gas

Chromatograph Series 5700A, Avondale, Pa., Decem-
ber, 1972, 3. With permission.
pounds. For example, the detector has a detection limit of 1 pg of lindane. Although
there are exceptions, the relative response factors of various functional groups to ECD
given in Table 3 can serve as a general guideline.
The electron source of an ECD may be either radioactive tritium or nickel-63. In
the tritium detector, the ratioactive material is either "adsorbed" or bonded or both
on a piece of stainless steel foil with titanium metal plated on one side and tritium
occluded therein. The titanium-tritium detector has an operation limit of 225°C to
prevent loss of tritium. This temperature is too low for many applications since detec-
tor contamination and decreased response may result from condensation of the sample
or column bleeding. Since the tritium cell offers less potential for thermal cleaning,
solvent washing is the only way to regain sensitivity and stability. In other designs
where scandium is used instead of titanium, the temperature limit is extended to 300°C.
This partially overcomes some of the disadvantages of the tritium detector as outlined
above. By contrast, the nickel-63 detector which has an upper operating temperature
of 350 to 400°C is more amenable to thermal cleaning. This optimum also means that
columns can be run at higher temperatures without the danger of condensation at the
detector. It is also noted that, for some compounds, higher detector temperature re-
sults in higher ~ e n s i t i v i t y Moreover,
.~~ nickel-63 has a half-life of 125 years, which is
about ten times that of tritium, and hence the nickel detector offers a longer usable
life than the tritium detector.
Neither pure helium nor argon are suitable carrier gases for the ECD since, in the
excited state, they may create metastable response by emitting an electron from a sam-
ple molecule which then gives a negative response to the recorder. This process is ef-
fectively eliminated by using nitrogen or a small amount of methane (5 to 10%) in
argon as carrier gas.
In the electron collection process, both tritium and nickel detectors can be operated
in the direct current (DC) or the pulsed mode.34In the DC mode, a continuous positive
voltage is applied t o the anode, while in the pulsed mode, the voltage is applied period-
ically. For DC operation, the detector sensitivity is a function of the applied voltage.
Different compounds require different voltages to give optimum response. Thus in the
case of a mixture, there will be a compromise in the choice of a proper voltage, and a
decrease in sensitivity for some compounds will result. On the other hand, iri the pulsed
operation, sensitivity is a function of pulse interval, and an optimum value can be
chosen for various compounds.
Another important characteristic of the ECD is that, although extremely sensitive,

the detector does not have the benefits of a wide linear dynamic range like that of the
flame ionization detector. Outside of this linear range, saturation of the cell occurs
and an increase in the amount injected does not yield a proportional increase in detec-
tor response. The ECD operated either in the DC or pulsed (constant frequency) mode
has a linear range of only 50 to 100, while in the pulsed (constant current) modes, the
linear range is improved to 103 to 104. Due to the relative nonlinearity of the ECD
response, standard curves are seldom used in O.C. analysis using EC-GLC. Instead,
the concentrations of the components in the sample are compared and calculated on
the basis of standards of comparable concentrations and chemical structures.
For the reasons cited above, the nickel-63 detector operated in the pulsed (constant
current) mode is the most desirable design at present. Further discussion on the char-
acteristics of ECD was given by L o ~ e l o c kand
~ ~ more
. ~ ~ recently by Aue and K a ~ i l a . ~ '
b. Alkali Flame Ionization Detector (AFID) and Nitrogen-Phosphorus Detector (N-PD)

The alkali flame ionization detector, also known as the thermionic detector, was
first introduced by Karmen and Giuffrida in 1964.38The principle of this detector is
based on the enhanced response of nitrogen- and phosphorus-containing compounds
in a hydrogen flame containing alkali metal atoms. An important part of the detector
is the alkali source; several volatile and nonvolatile salts such as chlorides, bromides
and sulfates of sodium, potassium, cesium, and rubidium as well as rubidium silicate
have been used. All of these salts show enhanced response and increased selectivity
towards phosphorus compounds. For the detection of nitrogen compounds, best re-
sponse and selectivity can be obtained when a rubidium salt is used. Theories have
been postulated to explain the ionization mechanism and the detection principles in-
~olved.~~.~~
Early designs of AFID have two basic disadvantages: (1) the lifetime of the alkali
source is short and (2) detector sensitivity does not remain constant and decreases as
the amount of alkali salt decreases. Many of these difficulties are related to the loss
of alkali salt in the hydrogen flame because of vaporization. A modification of the
AFID which is known as the nitrogen-phosphorus selective detector (N-PD) consists
of a burner jet, an alkali metal salt bead which is electrically heated (negative polarity),
and a collector electrode (positive polarity), with hydrogen and air flow rates much
lower than normal FID operation. Under these circumstances, the flame is not actually
ignited but it glows on the surface of the heated alkali source to produce dissociation
of ions. The advantages of this arrangement are (1) normal hydrocarbon ionization
reactions associated with the FID do not proceed efficiently and thus the detector pro-
vides high selectivity towards nitrogen- and phosphorus-containing compounds and (2)
there is no remarkable loss in the alkali source and thus the detector efficiency is main-
tained for a long time.
Many factors affect the response of the N-PD. These include carrier and detector
(hydrogen in particular) gas flow rates, alkali metal salt used, and potential across the
electrodes. Sensitivity and selectivity to a particular compound is achieved by optim-
izing the above parameters. Sensitivities for organophosphorus pesticides and triazine
herbicides are in the low nanogram range. Compounds with more than one nitrogen
per molecule (e.g., triazines) would be expected to show a proportionately greater re-
sponse than those with only one nitrogen per molecule (e.g., carbamates). Not all N-
containing compounds give good detector response. Nitro compounds are examples in
this class.
Since the N-PD is a highly sensitive detector, special attention and care must be
exerted in its applications in order to avoid anomalies.40 Glassware must be very clean
and free from phosphate detergent contamination. Silanizing reagents can decrease the
useful lifetime of alkali source so excess reagent should be removed prior to injection
Table 4
SPECIFICATIONS O F SOME G C DETECTORS USED IN PESTICIDE ANALYSIS
Detection Sensitivity Linear
Detector Selectivity limit range range Application
ECD(6')Ni) - <I pg of lindane W-Pg 1o4 o.c.s, PCBs, and

halogenated
compounds and
derivatives
N/P D 4 X lo4 g ~ / g ~ 10." gN/sec ng >lo4 o.p.s, triazines,
8 X 10' g c / g ~ 5 X 10-l4gP/sec carbamates
FPD 5 X 10' gC/gS 2 x 10-'l gS/sec W-Pg 10'-105 o.p.s, S compounds
10" gC/gP 10-I2gP/sec
Coulson loS ~ c / ~ N O.SngofCI,N;lng ng 10' o.c.s, Nand S
of S compounds
Hall 10" gC/gCl, N, S S x 10-l' g Cl/sec ng 10'-106 o.c.s, N a n d S
2-4 x 10-" gN/sec compounds
1-2 X 10." &/sec
MCD - 10-9g of Cl, N, and ng 10' o.c.s, N and S
S compounds
whenever possible. Phosphoric acid treated packings or glass wool, or nitrogen-con-

taining liquid phases (e.g., 0V-225, OV-275, XE-60, and the SP-2300 series) can add
noise to the system and should be avoided. The use of "Snoop" leak detector should
also be discouraged. A chlorinated solvent such as chloroform can give a large inverted
(negative) solvent peak and it temporarily affects the normal response of the detector
towards nitrogen and phosphorus compounds. Therefore the use of chloroform and
other chlorinated solvents as an injection solvent is not recommended. Care should be
taken to turn off the collector voltage while the detector gases are interrupted (i.e.,
during changing of the septum, column, gas cylinders, etc). Failure to do so will cause
overheating of the collector and thus deterioration of detector sensitivity.
Specifications and performance characteristics of the N-PD are given in Table 4.
c. Flame Photometric Detector (m)
The FPD is a highly sensitive and selective detector for sulfur and phosphorus com-
pounds. When a sulfur- or phosphorus-containing hydrocarbon compound is com-
busted in a hydrogen-rich flame, chemiluminescent species such as S, or H P 0 are
produced. By monitoring the characteristic emission maxima of these species (394 nm
for sulfur and 526 nm for phosphorus), detection and quantitation of sulfur- and/or
phosphorus-containing pesticides can be made in the nanogram to picogram range (see
Table 4). Because of the selectivity of the detector, samples require very little cleanup.
The response of F P D to phosphorus compounds is linear over a concentration range
of about 10". However, the square root of response to sulfur compounds is directly
proportional to the concentration. In other words, if the concentration is doubled, the
response increases by the square. It has also been found by Bowman and Beroza4' that
the relative response ratios of compounds containing phosphorus and sulfur are 5.2
to 6.0 for PS compounds, 2.8 to 3.3 for PS, compounds, and 1.7 to 2.3 for PS, com-
pounds. Therefore, the response ratio gives a measure of P-S content of the molecule.
The detector consists of a burner, a narrow band-pass optical filter, and a photo-
multiplying tube. Oxygen is mixed with carrier gas at the column outlet and then hy-
drogen is directly introduced into the burner without being premixed with the oxygen-
carrier mixture. The whole mixture is burned in a hollow tip which shields the flame
from direct view of the photomultiplier tube. Chemiluminescence which occurs above
the shielded flame is transmitted through the filter to the photomultiplier tube. An
improved version of this detector that permits simultaneous phosphorus and sulfur,
as well as flame ionization detection is also available.
d . Electrolytic Conductivity Detectors

Coulson detector - The original Coulson detector operates by combustion of GC
column effluent to form simple molecular species that readily ionize and contribute to
the electrolytic conductivity of deionized water. Thus nitrogen-containing pesticides
are coverted t o NH,, and organochlorines to HCl by reduction with H, over a nickel
catalyst in a pyrolyzer, and organosulfur compounds are converted to SO, and SO3
by oxidation in an oxygen stream. Changes in conductivity, which are directly propr-
tional t o the amounts of heteroatoms present, are monitored by a DC bridge circuit
and recorded on a recorder. This detector is specific to N, Cl, and S compounds and
detection can be operated at N, Cl, or S mode. Sensitivity is in the nanogram range of
N-, Cl-, or S-containing pesticides. Both selectivity and sensitivity in different modes
of operation are governed by furnace temperature, nature and flow rates of reactant
and carrier gases, flow rates of water through the cell, and choice of catalysts and
scrubbers.
Hall electrolytic conductivity detector (HECD) - The Hall detector3, is a modified
Coulson electrolytic conductivity detector with enhanced selectivity and sensitivity.
About tenfold increase in sensitivity towards nitrogen and chlorine compounds was
found for the Hall detector relative to the original Coulson detector. Detection limits
are at 0.1 t o 1 ng levels for polychlorinated insecticides (C1 mode) and 1 to 10 ng levels
for triazines and some nitrogen-containing 0.p.s (N mode). Hall has demonstrated that
the selectivity for aliphatic vs. aromatic chlorine may be realized by the proper choice
of furnace temperature, thus permitting selective determination of O.C.pesticides in
the presence of PCBs and PCNS.~'
For more details on the electrolytic conductivity detectors, the reader is referred to
the review given by Hall.,,
e. Microcoulometric Detector (MCD)

The MCD was the original detector designed for the detection and quantitation of
organochlorinated pesticides in the early 1960s. It operates by combusting column ef-
fluents to form HX (X = Cl, Br, or I), SO,, or NH, in a furnace and the reaction
products are titrated automatically with reagents electrochemically generated in the
coulometric cell. For example, in case of chlorinated pesticides, the Cl- formed is con-
tinuously titrated with the silver ions that are generated and kept at a constant concen-
tration by a reference silver electrode. Similarly, SO, is titrated with 1,- and NH, with
H+generated respectively by the I,/I- and platinum (hydrogen) electrodes. The amount
of ions generated is related to the absolute amounts of pesticides passing through the
G C column, and the peak areas can be converted to the quantity of pesticide present
by means of an equation without need for a calibration curve. At present, the MCD
is largely replaced by the HECD or by the ECD because of their higher sensitivity and
easier operation and maintenance procedures. Since the MCD is more specific (re-
sponds only to halogens, sulfur, phosphorus, and nitrogen compounds) than the ECD,
it requires less rigorous sample cleanup and can be used in the confirmation of organ-
ochlorinated pesticides. However, see discussion in Section VI.
111. PREPARATION OF STANDARD SOLUTIONS
Pesticide standards are conveniently prepared in a series of solutions of various con-

centrations commonly referred to as concentrated stock solution, intermediate concen-
tration stock solutions, and working standards.
A. Concentrated Stock Solutions

Using a 4-place analytical balance, weigh out exactly 25.0, 50.0, or 100.0 mg of the
"pure" standard in a weighing dish. If the purity is known to be less than 100% these
weights should be adjusted on the basis of its assay purity. Quantitatively transfer the
standard into a 50- or 100-m1 low actinic volumetric flask with a standard taper joint
by means of a small glass funnel. Rinse the dish and funnel carefully with solvent and
dilute to volume. Alternatively, if the volumetric flask can be placed on the weighing
pan of the balance, the pesticide standard can be weighed directly into the flask. Liquid
primary standards can be weighed into a 50-m! beaker, dissolved in a small amount
of solvent and transferred quantitatively by rinsing with the rest of the solvent through
the funnel into the volumetric flask. For highly viscous liquids, it is very difficult to
weigh exactly the required amounts. In those cases, attempts should be made to obtain
weights close to the required values and then only enough solvent should be added to
give exactly the desired concentrations. In all cases, no less than 10 mg of a standard
(preferrably 25 mg) should be weighed in order to minimize weighing errors.
Benzene dissolves most pesticide standards. Isoctane and hexane are alternatvies for
most organochlorinated pesticides; the former is preferred because of its low volatility
and hence less evaporative loss. Chloroform is useful for triazines and methylene chlo-
ride for carbamates.
B. Intermediate Concentration Stock Solutions

It is often necessary to prepare an intermediate concentration stock solution and
then the final working standard by dilution of the intermediate solution when highly
sensitive detectors such as the ECD and FPD are used in the detection and quantita-
tion. The preferrable dilution factor is between 1:100 and 1: 1000. Anything much
smaller than a 100-fold dilution may require yet another dilution before the working
standard can be made. Dilution much larger than 1000-fold may either require a large
amount of solvent which makes storage inconvenient or a very small (e.g., less than
10 PP) quantity of the concentrated stock solution which then grossly magnifies the
pipetting error.
If the stock solutions have been refrigerated, they should be brought to room tem-
perature before any pipetting is carried out. Volumetric pipets and syringes of the
exactly required volumes should be used whenever possible. Solvents such as isooctane,
hexane, or methylene chloride are commonly used for the dilution. Depending on the
situation, separate solutions of each pesticide or a mixture of all pesticides of interest
can be prepared.
C. Working Standards
These solutions are usually made up to concentrations suitable to the quantitation
techniques in use and/or to the expected levels of the pesticides in the sample extracts.
For the purpose of general screening or multi-residue analysis of environmental sam-
ples, working standards are usually made up as mixtures, provided the analytical tech-
nique has the capability to resolve them. In order to avoid nonlinearity problems of
some highly sensitive detector such as the ECD, the concentrations of the working
standards should closely resemble those in the samples when peak heights or peak areas
are compared. The solvents used in the preparation of working standard must be com-
patible with the quantitation technique in use and should be checked for any possible
interference.
All standard solutions of pesticides should be stored in an explosion-proof refriger-
ator at -4°C or below. It should be noted that benzene solutions can freeze at these
temperatures and may crack the container. Organochlorinated pesticide stock solutions
can be stored for at least 6 months without deterioration. Organophosphorus and car-
bamate solutions are less stable and should be discarded 3 ro 4 months after prepara-
tion.
A suggested concentration range for different pesticide solutions is listed in Table
5.
Table 5
CONCENTRATION RANGE O F PESTICIDE STANDARD
SOLUTIONS
Herbicide
OC OP Acid Carbamates Triazines
Conc. stock (mg/ml) l 1 1 1 1

Dilution one (pg/ml) 1-10 1-10 40-100 1-10 1-10
Dilution two (ng/ml) 10-100 - - -
1V. C O N F I R M A T I O N OF P E S T I C I D E I D E N T I T Y
In pesticide residue analysis, GLC with an appropriate detector is still the most
widely used technique for routine analysis. For the analysis of 0.c.s the almost univer-
sally used detector is the selective ECD because it provides the necessary sensitivity for
low level determination of 0.c.s in many applications particularly those in environmen-
tal surveillance and monitoring. Although the MCD in the chlorine mode can be more
specific than ECD, its lack of sensitivity for 0.c.s renders this detector useless in most
applications. Unlike a specific detector, ECD is selective in response to certain types
of compounds. Although electron-capturing compounds such as those containing hal-
ogen, nitrogen, oxygen, and aromatic moiety respond to ECD, the latter is far more
responsive t o polyhalogenated compounds. Thus, GLC-ECD is most suitable for trace
analysis of O.C. pesticides which contain several chlorine atoms. The other weaker elec-
tron-capturing compounds mainly derived from natural sources are often present in
the same sample as CO-extractivesand they are usually in thousands fold excess of the
0.c.s in question. Therefore, their ECD responses can be significant. In GLC analysis,
using any type of detector, the means of identifying unknown compounds is based on
the time required by the compound to traverse the column (expressed as retention time,
R,) rather than on a unique chemical characteristic such as in atomic absorption spec-
troscopy, polarography, UV or IR analysis. Consequently, identification based on a
single set of conditions is not valid; in fact, several compounds or CO-extractivesare
known to have similar GLC retention times (see Chapter 3). Therefore, additional
identification techniques to supplement initial GLC identification are necessary to pro-
vide additional substantiation to the identity of the pesticide in question. These suppli-
mentary identification techniques are often referred to as confirmatory tests. They can
be either physical or chemical or both. Generally, the approach to the confirmation
of identity comprises the following steps:
1. Tentative identification of pesticides based on GLC retention times on one GLC

column
2. Interpretation substantiated by GLC retention times from at least another col-
umn with different polarity (multi-column technique)
3. Other confirmatory tests:
a. Adsorption column elution pattern such as fractionation of pesticides by a
FlorisilB or silicic acid column
b. Thin-layer chromatography (TLC)
c. High-pressure liquid chromatography (HPLC)
d. Extraction p-value
e. Specific detectors
f. Spectroscopic and spectrometric analyses: nuclear magnetic resonance
(NMR), infrared (IR), mass spectrometry (MS), and ultra-violet (UV)
Volume I 51
g. Chemical confirmatory tests: wet (in solution), dry (in solid matrix), vapor
phase (reaction GLC)
h. Photochemical confirmatory test
The multi-column technique of using columns of different polarity to a certain de-

gree can be regarded as a confirmatory test if two or more suitable columns can be
chosen. Columns with nonpolar liquid phase such as OV-l or the semipolar column
of mixed phases (4% OV-101/6% OV-210) can be used in conjunction with a highly
polar column containing, i.e., DEGS. The correspondence between retention times on
a pair of columns of widely different polarity has been pointed out by Elgar,43quoting
the work of Thompson et to be less significant than the degree of concordance
between two different but more similar pair of columns. Thus, the degree of corre-
spondence of pesticide retention times between the intermediate polar OV-l/QF-1 and
OV-17/QF-l columns with respect to the nonpolar DC-200 column is highly signifi-
cant, whereas the degree of correspondence between the more polar QF-1 column and
the nonpolar DC-200 column shows less correlation. This indicates that QF-l is a better
column to provide additional substantiation of pesticide residue identity when used
with a nonpolar DC-200 column. The degree of concordance between a DC-200 col-
umn and a highly polar stationary phase DEGS column is even less significant. Con-
sequently, from the plot of relative retention times (to aldrin) of the investigated pes-
ticides on a DC-200 column and the columns mentioned above, Elgar43concluded that
"the highly-polar stationary phase DEGS makes an admirable compliment to the non
polar phases". It must be realized that although the degree of correspondence between
retention times on a highly polar and a nonpolar column is less significant, certain
concordance still exists and the degree of this concordance varies more or less from
pesticide to pesticide. Thus, this approach still cannot be considered positive because
as Robinson4' and R e y n o l d ~
pointed
~~ out separately, the multi-column GLC technique
cannot be regarded as an independent parameter of identity since it may be shown that
many pesticides on different stationary phases are significantly correlated. However,
the multi-column technique is a convenient and quick approach to provide additional
substantiation to the identity of a pesticide in question and can be quite positive in
some cases such as when the order of elution between compounds are reversed in the
polar phase column, e.g., oxons and parent 0.p.s on DEGS column (see Volume 11,
Chapter 2 on 0.p.s) or aldrin and lindane on an 0V-225 column (see Chapter 3) as
compared t o nonpolar or intermediate polar columns.
As in all approaches for the confirmation of pesticide identity, this multi-column
technique has another limitation. The mixture of pesticides usually encountered in sur-
veillance and monitoring work may not be as clearly separated in a single polar phase
column such as DEGS or 0V-225 as in intermediate polar mixed phase columns such
as OV- 17/QF- l , OV-1 /QF-l , or OV- 101/OV-225. Thus for some pesticides this multi-
column technique may not be applicable since they are not distinctly resolved in the
single phase polar column.
A. Adsorption Column Elution Pattern

In the analysis of o.c.s, almost invariably a FlorisilB, silicic acid, or an alumina
column is incorporated into the cleanup process before GLC examination of sample
extract. Due to the presence of other pesticides, industrial compounds, and co-extrac-
tives, graded elution is used to group the 0.c.s into several fractions (see Chapter 3,
Table 6 ; and Volume 11, Chapter 1). This simplifies gas chromatographic interpreta-
tion and is necessary in multi-residue analysis since some pesticides are not sufficiently
resolved in many commonly used GLC columns. The elution of a pesticide residue in
a certain fraction is a further indication of its identity. However, the use of adsorption
Table 6
EFFECT O F EXPOSURE O F
PESTICIDES T O MERCURY AND
COPPER
Percentage recovery
based o n mean of
duplicate tests
Compound Mercury Copper
BHC
Lindane
Heptachlor
Aldrin
Hept. epoxide
p,p'-DDE
Dieldrin
Endrin
DDT
Chlorobenzilate
AroclorC? 1254
Malathion, diazinon,
parathion, ethion,
trithion
column chromatography as a confirmatory test is not too specific as many co-extrac-

tives and compounds can be CO-elutedin a fraction with the pesticides in question.
For 0.p. pesticides, quite often column cleanup is not necessary (see Volume 11,
Chapter 2). The sample extracts, with or without liquid-liquid partitioning, are exam-
ined by GLC. Fractionation of the 0.p.s into different groups by column chromatog-
raphy and reexamination of the eluates can provide additional substantiation to their
identities.
Since many carbamates and all herbicidal acids (see Volume 11, Chapter 3 and Vol-
ume 111, Chapter 1) require derivatization of the parent compounds before GLC ex-
amination, the derivatives can similarly be fractionated into groups before GLC ex-
amination.
Triazine and some urea type pesticides can be analyzed without derivatization and
a similar approach such as that used for 0.c.s or 0.p.s can be contemplated. However,
in all cases, if adsorption column chromatography is not already incorporated into the
extraction and cleanup procedures, it is rarely used as a confirmatory test because it is
time consuming and less specific compared with other tests discussed here. Further-
more, the labile pesticides such as some 0.p.s and carbamates can be decomposed (on
the adsorption column) during column chromatography.
B. Thin-Layer Chromatography (TLC)

The limitation of TLC for confirmation of identity of pesticide residue is discussed
in Chapter 111, I.A.1. In short, it is not a specific procedure. In addition to its limited
resolution power to differentiate two closely eluted compounds with similar R, values,
this technique is subject to many variations, as was discussed previously in this chapter.
Some of these variations can be controlled although with inconvenience, but others
are difficult t o control.
Notably, even after cleanup procedures some CO-extractives are still present as
starchy or oily materials which may change the R, value of the compound in question,
making comparison with reference standard difficult or impossible.
C. High-Pressure Liquid Chromatography (HPLC)
Similar to gas chromatography, identification of a component in a liquid-chroma-
togram is made by its retention time or volume. Provided the sensitivity requirement
is met, one can further confirm the identity of a peak with a multiple or variable
wavelength UV detector by monitoring the analytical operation at two different wave-
lengths. If the peak height or area ratios of the standard and the sample at two differ-
ent wavelengths remain unchanged, one can be reasonably certain that the sample is
identical to the standard. The assumption is that it is highly unlikely to have two com-
pounds which have identical retention times and UV extinction coefficients at different
wavelengths at the same time.
The obvious limitation of this approach is that not all pesticides have sufficient UV
absorption. In particular, the O.C.pesticides have little or very low UV absorbance to
be considered by this approach. Also, the sensitivity of this technique for those parent
pesticides with UV absorbance is generally from microgram to nanogram levels and
can be less sensitive than the initial analysis by other approaches such as GLC with
specific detectors.
D. Extraction p-Values
Based on the distribution of pesticide between two immiscible phases the extraction
p - v a l ~ e ~ is
' - a~ ~powerful tool in identifying pesticides at the nanogram level. Each
pesticide has a characteristic distribution ratio defined as the p-value which is said to
be independent of pesticide concentration and the amount of CO-extractives.There are
several procedures to determine p-value: the simplest procedure is to distribute the
pesticide in question between two 5-m! portions of previously equilibrated, immiscible
phases in a glass-stoppered 15-m! centrifuge tube. A few microliters (e.g., 5 p 1 ) of a
pesticide solution in 5 m1 of nonpolar solvent (the nonpolar phase) is first analyzed
by EC-GLC or other means. Then 5 m! of the polar phase is added and the tube is
shaken for about 1 min. If necessary the tube is centrifuged to separate layers or break
emulsion. An equal volume of the nonpolar phase is then analyzed in exactly the same
way as before. The ratio of the second analysis to the first gives the p-value. This ratio
is the amount of pesticide in the nonpolar phase (second analysis) divided by the total
amount of pesticide (first analysis). Some of the typical solvent pairs are hexane/ace-
tonitrile, petroleum ether/acetonitrile and isooctane/acetone.
If sufficient aliquots are available, additional verification may be obtained from p-
values in other solvent systems. However, as Elgar43pointed out, the additional inde-
pendent evidence for identity gained by obtaining a p-value is very small.
This method works equally well when the sample contains a mixture of pesticides
provided they can be easily separated by the GLC column. The p-values for 136 pesti-
cides and six solvent systems have been reported by Beroza et al."
So far, all the physical confirmatory tests (multi-column techniques, adsorption col-
umn chromatography, TLC, and extraction p-value) are not considered as independent
parameters since as pointed out by Elgar,43 "the basis of separation of all these tech-
niques lies in partition phenomena, it would not be surprising to find some concord-
ance between values for a compound by all these techniques". Some examples are
illustrated in Section A of Chapter 3, notably a DDE artifact4s from certain plastics
having identical retention times in several GLC columns using ECD and MCD and
very close p-values.
E. Specific Detectors
The use of specific detectors for confirmation purposes is limited because they are
usually used for the initial analysis of pesticides. In certain cases where a less specific
detector is used for initial analysis, a more specific detector can be used for confirma-
tion. Sometimes a pesticide can be analyzed by one specific detector and its identity
confirmed by another; for example, an 0.p. containing both P and S atoms is initially
analyzed by FPD in the P mode and confirmed by the same detector in the S mode or
vice versa. Due to the difference in sensitivity and tolerance to sample CO-extractives
(see discussion on detectors and also Volume 11, Chapter 2, Section VII), this approach
is not always feasible. For O.C.compounds, although the electrolytic conductivity de-
tector in the chlorine mode is somewhat more selective than ECD, responses of this
detector as compared to ECD are so low that its usefulness is greatly limited.
F. Spectroscopic and Spectrometric Analysis

l. Nuclear Magnetic Resonance Spectroscopy (NMR)
NMR spectroscopy is a technique which provides structural information based on
the chemical shifts and spin-spin coupling patterns of a single compound. Proton NMR
spectra of organic compounds can be routinely run and interpreted by the average
operator. For compounds with no protons I3C NMR spectra can be run with a higher
degree of instrumentative and interpretative sophistication. Despite its merits, this
technique is rarely applied to pesticide analysis because of its poor sensitivity. Usually
a few milligrams of a single pure sample is required, although the CAT (computer
average of transients) and Fourier transform techniques which enhance signal-to-noise
ratios may cut the detection limit down to high microgram levels. This requirement is
unrealistic in residue analysis and the application of NMR is restricted to analysis of
pesticide standards only.
2. Infrared Spectroscopy (ZR)

IR spectroscopy is a popular technique used in obtaining structural, especially func-
tional group, information of organic compounds. It can easily be performed and the
spectrum obtained is the fingerprint of the compound. Standard IR spectra of most
common pesticides have been p ~ b l i s h e d . ~Sensitivity
' is at the microgram level. For
confirmation purposes, stringent cleanup of the sample by TLC, GLC, or other means
is required. Positive identification of organochlorine residues in sludge, soil, fish, and
industrial effluents has been reported,53 although the sensitivity requirement limits the
application to many environmental samples.
3. UV Spectroscopy
Similar discussion on HPLC with UV detector can be applied here. UV is useless
for the confirmation of o.c.s, o.p.s, and phenoxy acid herbicides on account of very
low or little UV absorbance. It can be used for some triazines and carbamates; in fact,
it is an alternative means for their initial analysis rather than by GLC (see Volume 111,
Chapters 1 and 3).
4. Mass Spectroscopy (MS)

This technique provides molecular weight and structural information of an organic
compound from the molecular ion and fragmentation pattern, respectively, of the de-
rived mass spectrum. Since the fragments are produced in a predictable and reprodu-
cible way, the mass spectrum is usually considered as the fingerprint of the compound
and is therefore useful in the confirmation of identity. The ionization process can be
done by either the electron ionization or chemical ionization technique. With the mass
spectrometer alone, a single pure compound is needed in order to obtain the above
information.
Up t o the present time, one of the most versatile and sensitive analytical techniques
for the pesticide chemist is the coupling of a mass spectrometer with a gas chromato-
graph and a computer system. Here the mass spectrometer is used as a "real time"
Volume I 55
detector for the gas chromatograph; both identification and quantitation can be estab-
lished on a complex pesticide residue mixture. Also, the mass spectrometer is the most
specific detector for G C by virtue of the confirmative information on molecular weight
and fragmentation pattern obtained. A recent review on the application of GC-MS to
pesticide analysis has been given.54
However, although quite versatile, GC-MS is not a panacea for confirmation pur-
poses in a routine laboratory. The pros and cons of using GC-MS as a confirmation
technique is discussed in detail in Chapter 3, Section V.
G. Chemical Confirmatory Tests

Chemical confirmatory tests refer to the technique whereby the pesticide after initial
analysis (usually by GLC) is reacted chemically or photochemically to form one or
more derivatives which are reexamined (usually by GLC). Since the derivatives are
different compounds from the corresponding parent pesticide, they exhibit different
analytical characteristics such as different GLC retention times or TLC R, values. The
use of derivatization in conjunction with multi-column technique is believed to be a
very specific approach indeed for positive confirmation of identity of a pesticide. De-
rivatization can be achieved by chemical reaction in solid, in solid matrix, and vapor
phase. Detailed discussion on these derivatization techniques are given in Chapter 3.
H. Photochemical Confirmatory Tests

The limitation and disadvantage of the photochemical derivatization technique for
confirmatory purposes has also been discussed in some detail in Volume 11, Chapter
2. Although it may be proven to be useful in a few cases, in general this approach is
less sensitive and convenient, generates more by-products, and is more subject to inter-
ference from CO-extractivesas compared to the chemical derivatization technique.
V. SOME ASPECTS OF QUALITY CONTROL
A. Introduction
It has been stated that nearly every phase of environmental protection and pollution
control depends on the ability t o identify and accurately measure specific pollutants
in the environment. For example, without reliable measurement, health officials can-
not correctly assess the health effects and relate them to levels of pollution; engineers
cannot correctly assess and hence improve the effectiveness of pollution control tech-
niques; regulatory agencies cannot correctly relate levels of pollution emissions with
ambient air and water quality; and the government cannot make appropriate decisions,
such as compromises among the conflicting demands of environmental protection, en-
ergy conservation, and economic health; and scientists cannot make suitable interpre-
tation on the environmental impact of pollutants, etc. Since "the analytical laboratory
provides qualitative and quantitative data for use in decision making"55 which has
such an extremely important financial, legal, or political implication, the ability to
identify and accurately measure specific pollutants in the environment is the most im-
portant consideration in pollution control, environmental protection and assessment
because "approximate or incorrect results are worse than no result at
Quality control activities are therefore the most important and essential tools to
insure the reliability of analytical data. The terms "quality control" and "quality as-
surance" have never been clearly defined and distinguished although these terms are
used quite often. Some authors imply that quality control activities are in-house activ-
ities to control quality of data, whereas a quality assurance program has a wider scope
to include interlaboratory quality control activities. However, these two terms are
often used i r ~ t e r c h a n g e a b l y . ~
According
~-~~ to the Handbook for Analytical Quality
Control in Water and Wastewater Laboratorie.P and Bretthauer et al.'' a quality as-
surance program implies a more formalized structure with a legal overtone to include
the evaluation and certification of laboratories as well as the development and issuance
of procedures such as the EPA quality assurance program. However, the H a n d b ~ o k ~ ~
also states that "quality assurance programs have two primary functions in the labo-
ratory", namely, (1) continual monitoring and reliability (accuracy and precision) of
the results reported and (2) controlling of quality. This implies that quality assurance
also includes activities inside the laboratory.
In our discussion, we are concerned with any activities that will affect the quality
of data and adopt Sherma'sz4 connotation of quality control in the context that it
connotes "procedures taken to assure the accuracy and precision of analytical results".
In this context of quality control to assure reliability of data generated, a good and
meaningful quality control program should encompass all the areas that could affect
the quality of the analytical data.
For practical convenience, a quality control program can be broadly divided into
two classifications, intralaboratory (in-house) Q.C. and interlaboratory (between lab)
Q.C. programs. Both areas complement each other and are necessary.
B. Purpose of Inter- and Intralaboratory Quality Control Programs

Among many other activities, the final step of an interlaboratory quality control
program involves the distribution of laboratory-fortified or naturally contaminated
samples to participating laboratories for analysis. The data are interpreted and usually
evaluated statistically by the coordinator of the program and a report sent to each of
the participating labs.
The purposes of the interlaboratory program could be
1. To assess and assure compatibility and quality of analytical data generated by

the participating laboratories, whether the same or different method for analysis
is used for the same parameter. In the case of different methods used, evaluation
on the compatability of these methods can also be the objective of the studies.
Compatibility of analytical data is particularly important in order to avoid em-
barrassment and waste of money if the data are pooled in the same data bank or
for a study to be used for scientific research or for government policy making.
2. T o identify analytical problems, particularly those usually not detected by in-
house quality control programs.
3. T o measure and provide statements on interlaboratory precision, accuracy, and
lowest detection levels so that data generated by contributing laboratories to the
data bank or for a study can be interpreted with demonstrated levels of confi-
dence.
4. T o detect training needs of laboratories in an organization.
5. T o evaluate and screen contracting laboratories and to monitor data quality of
the chosen laboratory.
6. T o monitor effectiveness and efficiences of the in-house quality control activities
of laboratories.
7. T o assist participating laboratories in overall upgrading of analytical perform-
ance.
The purpose of the intralaboratory quality control program could be
1. T o measure in-house precision, accuracy, and lowest detection levels of the anal-
ysis within the laboratory.
Volume I 57
2. T o detect analytical problems and immediately rectify them. This includes purity
problems of reagents and analytical standards, instrumentation performance,
laboratory contamination and so on.
3. T o detect weak methodologies.
4. T o provide a permanent record of instrument performance as a basis for validat-
ing data and projecting repair and replacement needs.
To be effective and meaningful, a quality control program is broader than normally

perceived. In the case of an interlaboratory quality control program, it is insufficient
merely to distribute samples to participants for analysis and generate a report after
the results are evaluated statistically. This is only the final step of a mechanical routine
in the program. This manner will not fulfil1 most of the objectives discussed above for
an interlaboratory quality control program and also will not provide the necessary
tools or guidelines for effective in-house quality control activities.
C. Structure of an Effective Quality Control Program

Therefore, a good and meaningful quality control program should control all the
areas that could affect the reliability and quality of data. The key areas that could
affect analytical data are
Sampling techniques (e.g., filtration or not; sample containers)

Sampling handling (e.g., preservation, storage)
Sub-sampling techniques (e.g., assurance of homogeneity)
Suitability of methodology (e.g., precision, accuracy, detection level, expedience,
"ruggedness" of method, chance of interferences)
In-house analytical practices (e.g., instrumentation calibration, trouble shooting,
purity of standards, storage of standards)
Confirmation of analytical identity
Suitable training of analysts and technicians
Availability and suitability of certified analytical standards to ensure right iden-
tity and suitable purity
Availability and suitability of standard reference materials for calibration of
methodology (including instruments)
Good laboratory practice
Control of all these activities should be an integral part of a quality control program.
Unfortunately, many of these aspects are not sufficiently emphasized or integrated as
they should be. An effective interlaboratory quality control program therefore should
have the following activities:
1. Maintaining a repository for analytical reference standards of pesticides, and

their major metabolite or degradation products and industrial chemicals and
after proper certification suppling them to routine laboratories
2. Validation, evaluation, and adaptation of analytical methodology to ensure that
the laboratories used the most suitable methods for the generation of analytical
data
3. Documentation and evaluation of various procedures used by laboratories con-
tributing data t o ensure compatibility of results
4. Investigation of procedures to prepare large bulk samples as standard reference
materials and prepare them for intra- and interlaboratory quality control studies
5. Investigation of and establishing guidelines and procedures for sampling, sample
handling, and preservation t o maintain integrity of analytical samples
6. Development of proper procedures (preferably with the laboratories in question)

for a proper intralaboratory quality control program
7. Provision of technical training for specific requirements and particularly on new
methodology
D. Discussion of Some Key Aspects of an Effective Interlaboratory Quality Control

Program
Some key activities in an effective interlaboratory quality control are discussed be-
low.
l. Analytical Reference Standards

"The greatest single source of quantitative error in GC analysis is undoubtedly in-
accurate standard solutions"58 and impure standards. Therefore, the purity and right
identity of analytical standards used must be ascertained. In pesticide residue analysis,
as well as in trace organic analysis, a satisfactory source of analytical reference stand-
ards has been a major concern for many laboratories. As pointed out by M c C ~ l l y , ~ ~
"most laboratories obtain their 'standards' from the manufacturers of pesticides and
d o not test (mainly due to lack of facility or experience to do so) the authenticity or
purity of these compounds. Some chemical supply houses offer a limited number of
'standards' but d o not provide data or any guarantee as to purity or authenticity of
the chemicals . . . .Residue laboratories also require standards of pesticide metabolites
since residues of these compounds must be determined."
Although the manufacturers are generally cooperative, it is a time consuming activ-
ity. To request and receive the "standards" a laboratory requires can be lengthy and
the quantity can be very limited. Sometimes, "pure standards" are not available.
Even if a laboratory can obtain all the required standards from different manufac-
turers, personal contact, or chemical supply houses, the purity is often not suitable
for trace analysis. There are many ways to label the purity of an organic compound,
e.g., by IR or UV spectrophotometry, titration (if acid or base), GLC, and so on. A
compound found to be, say, 99% pure by IR may be found to be less pure by GLC
analysis and vice versa.
Furthermore, many standards have unknown shelf stability. In the presence of some
impurity acting as catalyst or upon storage under non-ideal conditions, some classes
of pesticides such as 0.p.s or carbamates can undergo degradation. In some cases it is
difficult to detect the degradation during GLC analysis. For example, if a pure ester
is partially decomposed to a nonvolatile acid, GLC analysis will still show one ester
peak although the peak height will be smaller depending on the degree of decomposi-
tion. Naturally, the unaware analyst using this ester as analytical standard will generate
erroneous analytical results.
Obviously a central pesticide reference standards laboratory and repository is nec-
essary in an organization which consists of several laboratories engaging in similar
analysis. This laboratory and repository will ensure the availability, authenticity, pu-
rity, and proper storage of these standards by acquiring the standards, by synthesis of
the required standards not available commercially, and by proper in-house investiga-
tion and monitoring on their shelf stability and storage conditions. This laboratory
will distribute this information and the analytical standards, after certification of their
authenticity and purity, t o the analytical laboratories within the organization. By this
arrangement, time wasted by each laboratory to duplicate the collection, purification,
and preparation of a set of standards is eliminated, but most important of all, it will
reduce the chance of producing erroneous data and minimize misinterpretation by
eliminating contamination of the trace organic analytical laboratories if the necessary
purification and synthetic activities are carried out in these premises which are not
designed for macro scale work.
Volume 1 59
2. Suitability of Analytical Methodology and Compatibility of Analytical Data

Within an institution, the easier and most efficient way to get comparable data from
laboratories is the provision of uniform, standard analytical methods for these labo-
ratories. However, comparability of data does not necessarily mean the data are good.
A consistently poor method used by all laboratories can generate very comparable but
poor data. Therefore, methods must be thoroughly validated and evaluated before
they can be considered as standard methods. In order to avoid setting these methods
in concrete, not allowing for "state of the art" developments and improvements, they
should be constantly reviewed, revised, and improved by in-house investigation as well
as by interlaboratory comparisons. This responsibility should be given to a group
which develops, validates, and evaluates methods and also runs quality control so that
the developed or validated methods when in use by these laboratories can be under
periodical scrutiny in interlaboratory quality control studies. By proper designs of
these quality control studies, further information on the ruggedness or unrecognized
deficiency of the method can also be observed and any problems in methodology can
be quickly spotted and investigated. Thus it is necessary to integrate the activities of
method development and validation as well as quality control because the use of the
"best" available method is one of the fundamental steps to ensure the quality of data
generated.
Although the above integration of quality control and method development and
validation will ensure compatibility of data generated and enable action to be taken
on the improvement of the standard methods by appropriate design of quality control
studies, there is one major disadvantage in the approach of standardization of meth-
odology. The reason is that "a single standard method may cause a single type of error
to be in all data rather than just being a variable within a data set."59 This is easily
overcome by periodically running interlaboratory comparison involving laboratories
of other agencies using different methodology.
In some situations, laboratories that are supporting a surveillance program are mem-
bers of different agencies and institutions; they can be members of different levels of
government or even from different countries. Under these situations, standardization
of method is not feasible since it is difficult to have agreement to choose a single
method. It is also undesirable because, as mentioned, a single method may cause a
single type of error. Since so much effort and expense are put into a surveillance pro-
gram, compatability and data quality assurance are of utmost importance for the suc-
cess of the surveillance. Properly and suitably designed interlaboratory quality control
studies are needed to ensure that the data are valid.
In conclusion, it is desirable to have standardization of methodology among labo-
ratories within an institution t o ensure compatability of data. To avoid the pitfall of
not allowing for "state of the art" development and improvement, a group is needed
to improve and evaluate constantly these atandard methods and also to be able to
respond to analytical problems. Unnoticed bias of "standard" methods can be spotted
and method investigation can be readily initiated since method development and vali-
dation activities are integrated with qualtiy control studies in the same laboratory.
Thus validation, evaluation, and development of suitable methods is an integral part
of a quality control program for the assurance of data quality and compatability.
3. Standard Reference Materials (SRM)

Standard reference materials such as fortified and naturally contaminated waters,
sediments (wet or dried), or fish are invaluable and necessary in any quality control
activities. They can be used for the determination of precision within laboratory or
compatability between laboratories. But most important of all, SRM are needed for
the determination of accuracy of a method or the performance of a laboratory. They

are also invaluable for in-house calibration of methodology or instruments and provide
a quick on-the-spot checking.
In surveillance and monitoring activities quite often several laboratories contribute
data for the same study. The analytical results of a laboratory can be more meaningful
if they are accompanied by data obtained at the same time on SRM. Since these mate-
rials are identical to natural samples as in the case of using naturally contaminated
bulk sample t o prepare the SRM, their use to check out a developed or adapted new
method is much more meaningful and the evaluation much more realistic than tests
made on synthetic mixtures. Both the naturally contaminated or spiked standard ref-
erence materials can be used in-house by the analyst or in interlaboratory quality con-
trol studies to check and measure the performance capability of each analyst or each
laboratory, so that corrective actions can be taken by the individual before serious
problems occur and erroneous data are reported. The SRM is also invaluable in screen-
ing and checking contracting laboratories for performance capability and compliance.
Individually spiked samples always have the uncertainty that there may be mistakes in
spiking for certain batches of samples. Since the whole spiked sample has to be used
for extraction and analysis due to possible inhomogeneity, only certain spiked samples
in the series are checked. Those sent out to the participants cannot be checked. On
the other hand, SRM, spiked or naturally contaminated, are thoroughly mixed, tested,
and checked for homogeneity on a large number of subsamples. Thus, the problem of
erratic spiking is eliminated.
The SRMs are prepared in large quantities such as a few hundred kilograms or liters
so that they can be used periodically for a long period of time. However, to prepare
large quantities of these materials is much more complicated than it appears, since
there is little literature precedence particularly for organic parameters-for example,
investigation on how to ensure homogeneity of the sample, long-term storage condi-
tions to ensure integrity of the sample or subsamples, and in case of fortification, how
to distribute spiked compounds evenly and how to ensure no loss or degradation. In
addition to technical difficulties, t o prepare an SRM is also very time consuming be-
cause it requires collecting, freeze-drying (if necessary), and homogenizing a large bulk
sample as well as bottling, labeling, and cataloging of a few thousand subsamples. It
also requires several hundreds of analyses to determine levels of contaminants and
homogeneity and periodic monitoring of subsamples to ensure sample integrity.
4 . Sampling, Sample Handling, and Storage

The analytical data are only as good as the sampling techniques including handling,
storage, and preservation. In an area of trace organic analysis, in particular, these
aspects are far from established and they become one of the necessary activities to be
investigated in quality control design and planning.
Surprisingly, sampling, sample handling, and storage are not always regarded as the
responsibility of an analytical laboratory o r as one of the important aspects of a quality
control program. As stated in the H a n d b ~ o k , '"quality
~ control must begin with sam-
ple collection and must not end until the resulting data have been reported." It also
states that "quality assurance programs limited to the care of the sample beginning
with the receipt by the laboratory are inadequate. The laboratory must share respon-
sibility for the preservation and shipment of all samples that it will accredit with con-
centration values."
Sample handling, storage, and preservation techniques are also very important in
any quality control study because before quality control studies can be initiated, it is
necessary t o ascertain the integrity of the samples from the time they are collected until
they are analyzed. Stability of samples during storage and shipping must be maintained
to ensure the validity of the quality of the quality control study.
Volume I 61
T o develop procedures for sample handling and preservation requires tedious and
time-consuming investigation. For organic parameters, there is little literature prece-
dence t o be used as background information. Compounded by these problems, these
activities are generally not regarded by management as distinct projects or programs
in a laboratory operation; hence little or no manpower, time, and financial support
are allocated to them. It is even quite common (as seen in literature) that many re-
searchers who develop analytical methodologies often ignore the aspect of sample stor-
age, handling, or preservation. At best, the method includes some generalized recom-
mendation or brief preservation procedure usually without supporting data.
In regards to field sampling, there is usually very little dialogue between field staff
and laboratory personnel. Also lacking are field sampling and handling protocol,
which, if it existed, is usually described in general terms and not backed up with suffi-
cient data.
In summary, a quality control program to assure analytical data quality encompasses
several aspects rather than merely sending some sample out to the participants and
evaluating the results. T o be effective and meaningful, it involves all the aspects that
affect the reliability of analytical data. Thus provision and certification of analytical
reference standards, preparation and use of standard reference materials, development
of sample handling procedures, and validation, development and evaluation of analyt-
ical methods are necessarily integrated in an effective and meaningful quality control
program.
VI. DISCUSSION OF SOME KEY STEPS IN SAMPLE PREPARATION

FOR RESIDUE ANALYSIS
In pesticide residue analysis, there are many steps that could affect the quality of
analytical results. The most serious is perhaps caused by misinterpretation of the resi-
due identity. Misidentification of residue identity could be very expensive and embar-
rassing to a government or an institution if the data were involved in legal, interna-
tional agreement, or scientific research. Confirmation of residue identity is therefore
a very important aspect in the assurance of the analytical data generated.
Various approaches of confirmation of residue identity have been discussed in detail
throughout this book. In addition to misidentification, there are several steps that
could affect the quality of analytical data. The common ones are discussed below.
A. Evaporation
1. Evaporation to a Small Volume
Among the various manipulations involved in processing a sample for analysis (see
Section I1 of this chapter and Table 5 of Chapter 3), losses due to evaporation of a
sample extract have been greatly emphasized. For most pesticides, particularly the or-
ganochlorine pesticides and industrial chemicals such as PCBs, losses of compounds
at trace levels by improper evaporation are known. For some compounds, the method
of evaporation can affect significantly the degree of losses. As early as 1966, Burke et
investigated the evaporation of petroleum ether of five chlorinated pesticides.
"Various experiments with evaporation techniques and equipment have shown that
severe losses of pesticides may occur when solutions are concentrated to volumes of
0 . 5 m l or less." These authors found that "about 10 m! of solution can be rapidly
concentrated to 0.1 to 0 . 3 m l without loss of pesticide (0.c.s) by attaching a micro
Snyder column to a K-D collection tube and evaporating on a steam bath."
These authors also observed that:
1. Larger losses of pesticides occurred when solutions (petroleum ether) were evap-
orated to volumes of 0 . 5 m l and less by a gentle stream of air.
2. Losses increased as the residual volume approached dryness.

3. Percentage losses were greater when smaller amounts of pesticide were present.
4. Smaller losses resulted when the solvent (petroleum ether) was carefully boiled
off at steam bath temperature than at room temperature.
5. Oily materials such as those remaining after sample cleanup of certain samples
reduced evaporation losses.
Later, Chiba and Morley6' studied the losses of pesticides during sample preparation
which included evaporation. These authors found that evaporation of either benzene
or petroleum ether overnight in the fume hood at room temperature caused severe
losses of the three O.C. pesticides investigated. The losses of these 0.c.s from evapora-
ting a benzene solution to a small volume were usually considerably less than from a
petroleum ether solution.
These report^^^,^' indicate that evaporation of pesticide solution to 0.5 m1 can be
achieved by a Kuderna-Danish evaporator concentrator without loss of pesticides. Es-
sentially quantitative recoveries were obtained from evaporation of the same solution
using a gentle stream of air at room temperature in the presence of a suitable retaining
agent (oily substances or some sample CO-extractives). Without the retaining agent
(commonly referred to as keeper), considerable loss was observed.60
In the above-mentioned studies, the lowest concentration of pesticides investigated
was 1 pg in 7 m1 or 10 m1 (100 to 150 pg/p!) before evaporation. For water samples,
validation a t a lower level of pesticide concentration is needed since most natural water
samples contain low levels of pesticides. From experience, it was found that the prob-
lem of losses due to concentration of pesticide solution increases, though not linearly,
as the level of pesticides decreases. Therefore, an evaporation procedure which can
give satisfactory recovery at a higher level, say 1 pg/m1, may not give satisfactory
recovery at a lower level. Consequently, when we first engaged in water analysis in
1970, we investigated two commonly used evaporation procedures (K-D concentrator
and a gentle stream of air) at lower levels for all the 0.c.s we monitored in water
samples. Twelve 0.c.s were investigated in 1970; later the list was extended to 16 o.c.s.~'
Preliminary investigation on the evaporation of 10 m1 of hexane or benzene solutions
containing 13 0.c.s showed that if 0.5 m1 of toluene was added as a keeper there was
no significant difference on recoveries at 10 pg/pl-100 pg/p! levels by either the K-D
evaporative concentrator or by a gentle stream of air at 50 to 55°C water bath. Without
the keeper, however, the K-D method as described by Burke et gave a better
recovery. Later, purified nitrogen was used instead of laboratory air to avoid possible
contamination from the air line and possible degradation of more labile pesticides such
as 0.p.s (see Volume 11, Chapter 2, Section VIII). Our choice of toluene as a keeper
was derived from the observation that evaporation of a 10-m! pesticide solution in
benzene without keeper at 50 to 55°C to 0.5 m! with NZ gave better recoveries even
for the more volatile 0.c.s (e.g., HCB, lindane) than the evaporation of a correspond-
ing hexane solution under the same conditions. It was thought that toluene having an
aromatic moiety as the DDT group and a higher boiling point would serve as a good
keeper. In fact, a t the low level we investigated, it was found that on the whole toluene
is a somewhat better keeper than Nujol (10, 20, and 30% Nujol in 10-m1 pesticide
solution), or isooctane. In our investigation of using a gentle stream of N2 to evaporate
10 m1 of 16 0.c.s pesticides solution at 10 to 100 pg/p! levels to 0.5 m!, we observed
the following:
1. In the presence of a keeper, evaporation of the solution at an elevated tempera-

ture (50 to 55°C) gave a considerably better recovery than at room temperature
under the same conditions.
Volume 1 63
2. In the absence of a keeper, evaporation of pesticide solution at room temperature

gave higher recoveries than at elevated temperatures.
3. Without keeper, recoveries from evaporation of a benzene solution were higher
than from a hexane solution under the same conditions.
4. Recoveries varied with pesticide concentration; the higher the concentrations, the
higher the recoveries, even under less ideal conditions.
5. Among the keepers tested, toluene (0.5 to 1 mP) in 10-ml pesticide solution gave
better recoveries (almost quantitative) than isooctane or mineral oil under the
same conditions.
6. Even in the presence of toluene as keeper, evaporation below 0.4 to 0.5 m! is
not recommended for most pesticides.
7. Under no circumstances should the O.C. pesticide solution be evaporated to dry-
ness, perhaps with the exception of very oily sample extracts (e.g., fish).
T o conclude, evaporation of O.C.pesticide solution can be carried out by using a K-

D evaporative concentrator as described by Burke et or by a stream of nitrogen
at 50 to 55' C. For more labile pesticides such as some 0.p.s. and carbamates, the
"nitrogen method" is advantageous to avoid possible thermal degradation or oxida-
tion. It has been shown that the "nitrogen evaporation method" is suitable for ~ . p . s , ~ ~
car barn ate^,^^ and phenoxyacid ester^.^',^^ For phenoxylakanoic acids, there is no prob-
lem in evaporation at atmospheric pressure even when organic solutions containing
these acids are evaporated to dryness.
As a general method which has been tested for a large range of pesticides, the "ni-
trogen evaporation method" is recommended as an alternative procedure to the K-D
evaporation method. The "nitrogen method" is described as follows: Add 1 to 2 m1
toluene to sample extract and reduce volume by a rotary evaporator to 1 to 2 m! at
35 to 40°C. (For details see the next section.) Transfer, with hexane rinsings, the con-
centrated extract to a 15-mP graduated centrifuge tube (total volume at or below 10
mP). Add 1/2 mP toluene and evaporate extract under a gentle stream of pure nitrogen
at a 50 to 55°C water bath temperature to 0.5 m l . Do not evaporate extract below 0.5
mP .
Remarks:
1. In transferring the concentrated extract (1 to 2 m!) from the round bottom flask
to the centrifuge tube, transfer as much extract as possible before addition of
fresh solvent. Rinse the inside of the disposable pipet with no more than 1 m!
of hexane into the flask. Swirl flask thoroughly to rinse the inside glass surface.
Transfer rinsing with the same disposable pipet into the above centrifuge tube.
Repeat the process several times with 1 mP of hexane each time until the total
volume of solution in the centrifuge tube is about 9 to 10ml. These careful rins-
ings are necessary to transfer the solution quantitatively to the centrifuge tube.
2. Centrifuge tubes with a tapered end should be used to minimize inaccuracy of
reading 0.5 mP. The tubes can be precalibrated to check the accuracy of the
manufacturer's calibrated marks at 0.5 m! by putting 0.5 m1 (500 p! syringe)
solvent into the dried tube.
3. It is imperative with any concentration methods (K-D or nitrogen) that after con-
centration to the desired volume (0.5 m1 in this discussion), the tubes be rotated
to wash down the sides of the tubes to ensure a homogeneous solution and repro-
ducible, quantitative results. Some analysts in our laboratories rinse the side of
the tubes with 1 mP or so of fresh solvent (hexane or petroleum ether) during
concentration by nitrogen (e.g., when extract is concentrated to 2 or 3 mL). After
concentration to 0.5 mP, the tube is rotated so that the concentrate wash the
sides of the tube at 4 to 5 m1 mark rather than at the level of the original solvent
prior to evaporation. This will avoid exposure of a small volume to a large sur-
face.
4. Stopper tubes immediately after concentration to minimize evaporation. Exam-
ine concentrate by GLC as soon as possible. If immediate examination is not
possible, store stoppered tubes in a refrigerator until analysis. To prevent exces-
sive evaporation, wrap a thin film of TeflonB around the glass stopper and twist
tightly. Examine and adjust volume of each tube before GLC analysis.
2. Evaporation by Rotary Evaporator

In the above discussion, we have discussed the critical step of reducing several milli-
liters of concentrate to a very small volume (0.5 mP) for GLC analysis. When a sample
is extracted and/or cleaned up, the volume of the extract or cleanup extract can
exceed 100 mP of organic solvent. This volume is conveniently and usually reduced to
several milliliters by a rotary evaporator before further reduction of the volume to 0.5
mP or 1 mP as described above. Reduction of 100 m1 or more of a pesticide solution
to several milliliters is less critical than evaporation to 1 to 0.5 m l . No keeper is nec-
essary. However, if reducing an organic extract to only 1 to 2 m1 by a rotary evapo-
rator (under reduced pressure) certain precautions should be noted:
1. Add 1 to 2 mP toluene as a keeper before evaporation.

2. Use a water bath temperature of 35 to 40°C to evaporate solvents with boiling
points under 100°C. When the volume of the solvent reaches 8 to 10 m! remove
from water bath and let evaporation continue at room temperature to 1 to 2 mP.
3. Never evaporate to a volume below 1 mP in a rotary evaporator.
Almost all pesticide residue methods use organic solvents with low boiling points
(below 100°C) for extraction, partitioning, or elution from adsorption chromatogra-
phy. Those few methods that use a higher boiling point solvent such as acetonitrile
for extraction can be replaced by a solvent with lower boiling point by liquid-liquid
partitioning. Volume reduction of several hundred milliliters of higher boiling point
solvent by evaporation is not only time consuming but also increases thermal degra-
dation or oxidation of some pesticides.
B. Replacement of One Solvent by Another

Replacement of one type of solvent in a sample extract by another type is a com-
monly encountered practice in many residue analytical procedures. There are several
occasions when this practice is needed. For example, when the sample extract is in a
solvent which is incompatible with the detector such as halogenated solvent and ECD,
this detector-incompatible solvent must be replaced by a compatible solvent such as
hexane, benzene, or isooctane. In adsorption chromatography, column elution often
begins with a nonpolar or less polar solvent, followed by intermediate polarity sol-
vents, and finally with more polar solvents. For example, the commonly used FlorisilB
column cleanup and fractionation procedure for 0.c.s involves elution of column with
hexane or petroleum ether (nonpolar solvent) followed by solvents of increasing polar-
ity such as 6% diethyl ether in hexane (intermediate), 25% diethyl ether in hexane,
and sometimes finished with 50% diethyl ether in hexane or with chloroform. If the
sample extract to be chromatographed is in a more polar solvent than the solvent used
for the first elution, then the solvent in the sample extract must be replaced by a less
polar solvent or the same solvent as the first eluting solvent. Therefore in PCBs and
O.C. analysis if the sample extract is in methylene chloride then it is replaced by hexane
or petroleum ether before putting onto the FlorisilB column.
Failure in the removal of small amounts of polar solvent in the extract can alter the
column elution pattern of the pesticides. Its presence is very critical, particularly in
minicolumn application. There are three general methods for replacing solvents. They
are described below.
I. For a Large Volume of Solvent

Several hundred milliliters of a solvent (boiling points below 100°C) is replaced by
another solvent by first concentrating the original solution to 5 to 10 m l in a rotary
evaporator as described earlier. To this, 100 to 200 m1 of the desired solvent is added
and the solution is again concentrated to 5 to 10 mL. This process can be repeated to
ensure complete replacement. This process has been found satisfactory for replacing a
lower boiling solvent with a somewhat higher boiling solvent such as by replacing meth-
ylene chloride by hexane. (There is no problem in replacing a low boiling solvent such
as diethyl ether with a higher boiling solvent such as benzene.) However, if the original
solvent has a significantly high boiling point such as does acetonitrile, it cannot be
replaced by this evaporation technique. Liquid-liquid partitioning is used in this case.
Although no detailed studies are published on the replaceability of one solvent for
another by this evaporation technique, many procedures and the experience of many
residue analysts, including ours, indicate that this technique is effective. As a general
guideline, solvents with 50 to 80°C boiling points are easily replaceable by this tech-
nique. Hexane, petroleum ether, benzene, acetone, methylene chloride, and chloro-
form are solvents commonly used for sample extractions and they can be easily re-
placed by each other by this technique. To replace diethyl ether by either one of the
above-mentioned solvents is very easy since diethyl ether is very volatile. However, the
reverse is difficult. Fortunately there is no procedure in residue analysis that we are
aware of which requires the replacement of these solvents with diethyl ether.
2. For a Small Volume of Solvent

A few milliliters of one solvent is replaced by another by first concentrating the
original solvent down to 1 to 2 m l at 50 to 60°C with a gentle stream of N, as de-
scribed. Toluene cannot be used as a keeper in certain cases because of its intermediate
polarity. Isooctane can be used under this circumstance: 4 to 5 m l of the desired sol-
vent is added and evaporation is repeated. The effect of this evaporation technique is
generally not as effective as the rotary evaporation technique. Therefore, two or three
repetitive evaporations are required. An example of using this technique occurs in the
analytical procedure for several N-methyl carbamateP4 by derivatization of the hydro-
lyzed phenols or thiophenols with pentafluorobenzyl (PFB) bromide. Since this reac-
tion is carried out in acetone, this solvent must be replaced by a less polar solvent
before the reaction mixture is cleaned up and fractionated on a mini-silica gel column.
Thus, the PFB reaction mixture in acetone is evaporated with 2 m1 isooctane to 1 m l
in a 35 to 40°C water bath with N, gas. Evaporation is repeated once before the l-m!
concentrate is put on a silica gel mini column pre-wet with hexane before elution with
5% benzene in hexane in order to remove excess PFB reagent. Poor fractionation of
the PFB derivatives is observed when traces of acetone are not removed completely.
3. For High Boiling Point Solvents

As mentioned above, for solvents with higher boiling points such as acetonitrile,
replacement with a considerably lower boiling point solvent such as hexane or even
benzene is difficult or not feasible by the evaporation methods discussed above. For-
tunately the high boiling point solvents used in pesticide residue analysis are mostly
limited to acetonitrile, ethanol, dimethyl formamide, and dimethyl sulfoxide, and they
are all water soluble. Among these solvents, acetonitrile is one of the most frequently
used solvents in pesticide residue analysis. Due to their miscibility with water, sample
extract in these solvents can be easily replaced by partitioning with a lower boiling
point solvent such as hexane, benzene, cyclohexane, methylene chloride, or chloroform
in the presence of water. For certain pesticides such as 0.p.s. (see Volume 11, Chapter
2) and carbamates (see Volume 111, Chapter l), acetonitrile is a popular extraction
solvent for solid samples. It is also sometimes used for the extraction of some 0.c.s
from solid samples. The following example illustrates the replacement of acetonitrile
with a lower boiling point solvent such as petroleum ether.
An example is the nonfatty product procedure, 29.009(a) of the U.S. Pesticide An-
alytical M a n ~ a l , ~
which
' uses acetonitrile as the extracting solvent. Acetonitrile is re-
placed by petroleum ether by diluting the extract with water and, then extracting with
petroleum ether.
Another example of similar "flooding-out" of the pesticides and PCBs into hexane
or petroleum ether for the analysis of 0.c.s and PCBs in fish and sediment is described
in the Inland Waters Directorate Analytical Methods Manual. 68
C. Removal of Water in Water Immiscible Solvents.

Water immiscible solvents such as petroleum ether, hexane, benzene, chloroform,
methylene chloride, isooctane, ethyl acetate, and diethyl ether are commonly used for
extraction of pesticides and organic contaminants from solid (sediment or fish) and
liquid (water or wastewater) samples. Although they are more or less immiscible with
water, traces of water are present in the sample extracts if the samples contain water.
Some solvent such as diethyl ether and ethyl acetate dissolve significant amounts of
water. Traces of water must be removed from the sample extracts if they are to be
cleaned up by column chromatography or analyzed by GLC since the presence of water
is undesirable. In adsorption column chromatography, the presence of water will affect
effective fractionation of pesticides or other organic contaminants by lowering the
adsorbent activity. In GLC analysis, traces of water are generally thought to decrease
GLC column life.69
Anhydrous sodium sulfate is generally used to remove small amounts of water in
the sample extracts. Although it is not the most efficient dehydrating agent, its popu-
larity may be attributed to the availability of several particle sizes ranging from gran-
ular to powder. The granular form facilitates filtration of sample extract. However, if
the granules are too large, the water-removing capacity will decrease due to less surface
areas available. A good compromise is a somewhat coarsely powdered form. We have
been using this form of anhydrous sodium sulfate for several years to remove small
amounts of water in biota (e.g., fish), for o.p.s, carbamates, triazine, urea type herbi-
cides and neutral organic compounds such as PCBs. For acidic compounds, particu-
larly the phenoxyalkanoic acids or carboxylic acids, anhydrous sodium sulfate is un-
desirable since some of these acids will be adsorbed, causing a detrimental effect on
For c h l ~ r o f o r m or
' ~ methylene chloride7' extract, filtering through a small
plug of cotton will remove all the water in the extract. This technique may not be
applicable to ethyl acetate or diethyl ether extracts because these solvents dissolve con-
siderable amounts of water. Needless to say, the cotton must be adequately washed
with solvent, dried, and checked before use to avoid possible contamination of the
sample.
A more general drying agent for acidic compounds is the "acidified anhydrous so-
dium sulfate" described by Goerlitz and Lamar72which is summarized below for the
reader's convenience.
1. Reagent grade, anhydrous, granular sodium sulfate is heat treated at 300°C for
24 hr.
Authors' Note 1 - Due to the tendency of the acidic compounds to be ad-
sorbed, powder sodium sulfate should be used with caution since it has not been
tested to find whether the acidification treatment is also effective for the powder
form.
Note 2 - Heating at 300°C is used mainly to remove possible interferences.
As mentioned elsewhere, heating alone is not always effective. Washing with sol-
vents, air drying and then heating at 300°C is recommended for more severely
contaminated sodium sulfate.
Note 3 - After heating, some batches of sodium sulfate may change to a
greyish color. This usually does not affect its usage and the grayish color does
not cause interference.
2. The heat-treated material is cooled in a desiccator to room temperture, slurried
with enough pesticide grade diethyl ether to cover the crystals, and acidified to
p H 4 by adding a few drops of pure sulfuric acid.
Note 4 - Sulfuric acid should be checked against interferences by diluting
with water (CARE!) to at least the same amount of acid one would expect from
using the sodium sulfate as drying agent. Extract the solution with the solvent
used for the sample, and carry out the procedure for sample analysis.
3. T o determine the pH, a small quantity of the slurry is removed, the ether is
evaporated, water is added to cover the crystals, and the p H is measured on a
p H meter.
Note 5 - It is not certain whether it is necessary to have an exact pH of 4 or
not. Generally a lower p H is desirable to avoid or minimize adsorption. In fact
it is the authors' opinion that a p H lower than 4 is desirable. In any event, pH
can be more conveniently determined by just dissolving some slurry with distilled
water and checking the p H of the aqueous phase with narrow range pH paper.
It is not necessary to evaporate off the ether in this case.
4. After attaining the proper pH, the ether is removed by vacuum (rotary evapora-
tor) from the "acidified sodium sulfate" which is stored at 130°C.
In the early 1970s, we had the opportunity of evaluating the application of this
"acidified sodium sulfate" for removing traces of water in benzene extracts of water
samples.'l At the low ppb levels of the phenoxyacetic acid herbicides we found, that
the recoveries varied. On eliminating the use of acidic sodium sulfate, the precision
improved. This could be due to different batches of any impurity in sodium sulfate
affecting the result. Nevertheless, t o avoid unforeseeable problems, we chose to use
azeotropic evaporation to remove traces of water in organic extracts.
Since benzene and water form an azeotropic mixture, benzene is a commonly used
solvent for azeotropic evaporation to remove traces of water. A sample extract in a
water immiscible solvent is concentrated to 2 t o 3 m l in a rotary evaporator at 35 to
40°C as described previously. To this, 100 to 200 m l benzene is added and the mixture
is again concentrated to 2 to 3 m l . This process can be repeated once or twice if nec-
essary. U p to 2 to 3 m! of water in a sample extract can be removed this way. For
phenoxyalkanoic acids, a higher evaporation temperature (40 to 50°C) can be used,
since they are nonvolatile as compared to 0.c.s and 0.p.s.
D. Special Treatment of Glassware

For o.c.s, o.p.s, carbamates, and triazine compounds, the normal laboratory prac-
tice used t o wash the glassware is sufficient. That is, after the usual acid or alkaline
(detergent) washing, the glassware is weil rinsed with water to remove traces of acidic
or alkalinic substances before rinsing well with pesticide grade solvents.
However, for the acid herbicides such as phenoxylakanoic acids, particularly at trace
levels, special treatment of glassware is necessary to prevent adsorption of the acids

on the glass surface. This is easily achieved by rinsing clean glassware shortly before
use with diluted hydrochloric acid (l:3),followed by a few rinsings with distilled water.
Water miscible solvent such as acetone can be used to rinse out the glassware to facili-
tate drying at room temperature. This acid treatment is thought t o "neutralize" the
"alkaline" glass surface to avoid the adsorption of the acid herbicides onto it.
E. Solvent Purity
The purity of solvents used in analysis is undoubtedly an important aspect for the
success of the analysis and the reliability of the data generated. Generally there are
two types of solvent impurity. One is immediately detectable during the analysis by
the presence of a large solvent front or unusual extraneous peaks in the chromatograms
of sample extracts or standard solutions prepared by using these "impure solvents".
An alert analyst will usually spot this problem immediately, upon examination of the
chromatograms. It may be pointed out that pesticide grade solvents are not immune
to these interfering problems.
The other type of solvent impurity is more difficult to suspect and detect. It is not
the presence of extraneous peaks or a large solvent front in the chromatograms, but
rather the presence of other minor or trace constituents which are undetected by the
detector used. Therefore, although the "solvent test" may not show any background
peaks in the chromatogram, this solvent could contain impurities causing problems in
the analyses. One example is the presence of aromatic or conjugated hydrocarbons in
hexane. Even after hundred-folds of concentration, injection of this hexane into a
GLC using ECD may not shown any background interference. Yet when this hexane
is used as the eluting solvent in column chromatography, channeling of some pesticides
or PCBs into the unexpected fraction may occur, causing confusion or unsuccessful
fractionation. Z i t k ~ and
' ~ Bevenue et have respectively documented this problem.
Another example is the pesticide grade ethanol. Although it shows no ECD peaks, it
contains other chemicals used in denaturation of ethanol. If this denatured pesticide
grade ethanol is used in certain applications, it could be undesirable. For example,
preparation of a potassium or sodium hydroxide solution using this denatured ethanol
could generate ECD responsive compounds caused by alkali condensation of the ke-
tone or aldehydes used in some formulation of denatured alcohol.
Yet another example is the presence of impurities, believed to be peroxides, in diethyl
ether, which seriously alters the elution of pesticides from the FlorisilB column. Die-
thy1 ether is a solvent used in the FlorisilB column cleanup and fractionation proce-
dures (see Chapter 3, Table 6 and Volume 11, Chapter 1 on 0.c.s). Peroxides, being
polar compounds, would cause some overlapping of pesticides in the fraction. It may
also be pointed out that some pesticide grade ether contains 2% or so of ethanol as a
preservative to avoid the formation of explosive peroxides. It is also undesirable to
use this ether in the eluting solvent mixture for adsorption column fractionation. An-
hydrous diethyl ether does not contain this perservative.
In the case of hexane, several researchers, including us, observed that GLC/ECD
examination of a freshly opened pesticide grade hexane may show little or no back-
ground peaks and a very sharp solvent front in the chromatograms. However, upon
standing for some time in the laboratory, reexamination of this hexane shows a large
solvent front; thus, the size increases with time of storage. In 1970, we first observed
this phenomenon and one of our colleagues in the laboratory attempted without suc-
cess to purify this hexane by several fractional distillations using a fractional distilla-
tion column packed with more than 30 cm of tiny glass beads. It was by passage of
the redistilled hexane through several hundred grams of activated alumina that these
impurities were removed. Another more vigorous purification procedure published by
W i l l i a m ~ 'also
~ deals with this hexane problem.
Volume I 69
With the advent of pesticide grade hexane we are still experiencing this problem
periodically. All of the hexane has been obtained from the same local manufacturer.
Consequently, we use petroleum ether in place of hexane to avoid this problem or
obtain hexane from another manufacturer.
F. Emulsion
Emulsion during liquid-liquid extraction is commonly encountered by analysts. The
varieties of sample types and different qualities and quantities of CO-extractivesin the
same type of sample can cause different degrees of emulsion. The type of organic
solvents used in the liquid-liquid partitioning can also affect the degree of emulsion.
Most open waters such as the Great Lakes would generate little emulsion when water
samples from these sources are extracted with organic solvents. In agricultural run-
off, sewage effluent, and some industrial wastes, usually considerable emulsion is en-
countered. It appears that water samples containing sufficient particulate matter or
humic organic substances would cause emulsion.
After extraction of these water samples with a water immiscible solvent such as hex-
ane, benzene, or methylene chloride, the emulsion can be broken or decreased by either
one or a combination of the following methods:
1. Add several drops of saturated sodium sulfate solution and gently agitate the
layers to break emulsion.
2. Add a few grains of anhydrous sodium sulfate crystals and agitate layers gently.
3. Add a few drops of ethanol, isopropyl alcohol, butanol or octanol.
4. Use a piece of clean copper wire with a few flat loops at the end, moving the
loops up and down gently between the emulsified layer for a minute or two.
5. In a severe case of emulsion when one or more of the above steps is not satisfac-
tory, transfer and combine the organic extract with emulsion to a clean separa-
tory funnel, add distilled water approximately 3 times the volume of combined
organic extracts, and tumble contents very gently two or three times. Let phases
separate. If necessary a few grains of anhydrous sodium sulfate crystals can be
added.
6. Pass emulsion and organic extracts through a medium-packed glasswool column
(e.g., 2- X 3-in. glasswool) by gravity force and collect the separated layers in a
separatory funnel.
7. Centrifuge emulsion t o separate layers. This technique is very efficient but limited
to smaller total volume due to the size of the centrifuge available.
Such a severe emulsion problem is less frequently encountered in the extraction of

water samples. When organic extracts of sediment, fish, or plant material are required
t o partition with an aqueous phase, particularly an alkaline one, as in some procedures,
emulsion is usually encountered. Solvents which are somewhat soluble in water such
as benzene, ethyl acetate, and diethyl ether appear to have a slightly greater tendency
to form emulsions than the less water soluble solvents such as hexane or petroleum
ether. An acidic aqueous phase also has a lower tendency to form emulsions with the
same solvent than a neutral or alkali phase. Oily sample extracts such as those from
fish or animal tissue have a greater tendency to form emulsions particularly when they
are partitioned with an aqueous alkaline solution. It is also observed that acidification
of the sample before or after extraction decreases the tendency of emulsion formation.
G . Sulfur Removal
In certain types of samples such as sediments from areas of high biological activity,
the presence of sulfur and sulfur compounds is commonly encountered. Sulfur and
sulfur compounds can cause interferences to the analysis of aldrin and other O.C.pes-
ticides. In the presence of large amounts of sulfur or simple sulfur compounds a large
solvent front can occur, masking the early eluting O.C. peaks auch as lindane, hepta-
chlor, or even heptachlor epoxide. Sulfur compounds can also "poison" the ECD.
Therefore, in the analysis of 0.c.s in sediment, sewage sludge, and samples containing
sulfur or simple sulfur compounds, it is necessary to remove them or minimize their
concentration t o avoid interference t o the analysis and t o prolong detector life if ECD
is used.
The presence of sulfur will also be evident in the FPD and Coulson electrolyic con-
ductivity detector. The most popular method is to treat the sample or sample extract
with activated copper or elemental m e r ~ u r y . ' ~
This
, ~ ~has been discussed in Vol-
ume 11, Chapter 1 on 0.c.s. C ~ m p a r i s o n 'of ~ the effect of Cu dust and elemental mer-
cury on some pesticides indicates that Cu dust is generally more desirable. This com-
parison is reproduced in Table 6.
In addition to the degradative nature of elemental mercury, it also has the disadvan-
tage of being less efficient in the removal of of sulfur from the sample extract. Once
the surface of the mercury droplet is reacted with sulfur to form the black mercuric
sulfide, further reaction is stopped. Vigorous vibration will produce a more "active"
surface by breaking up the droplet. However, in some sediment extracts containing
high sulfur content, several treatments of the same extract with fresh mercury are re-
quired. Each treatment requires transferring the extract to another tube to be exposed
to fresh mercury. Obviously this is very inconvenient. In certain sediment extracts such
as those of Hamilton Bay in Ontario, Canada three or more treatments with fresh
mercury droplets are required t o remove the sulfur as evidenced by persistence of the
size of the solvent front after one or two treatments.
In contrast, activated dust has much more surface area to react with sulfur and
hence is more efficient than mercury. Also, additional copper dust, if required, can
be added directly into the same tube containing used copper dust without affecting the
sulfur removal efficiency since each copper particle exists individually.
It is suspected that some simple sulfur compounds are also removed by either mer-
cury or activated copper dust because some peaks in the regions between the solvent
and aldrin are also removed. The major sulfur peak has retention time close to or
identical with aldrin. Sulfur can have two or three peaks depending on source and
batch. These could be attributed to the different crystalline forms of sulfur.
In using either mercury or copper for O.C. analysis, one should be aware of its limi-
tation in that heptachlor is degraded drastically after treatment (see previous table).
Previous analysis of several hundreds of Great Lake sediments failed to reveal any
heptachlor. In a few cases of its suspected presence, the levels were so low that
confirmation was not conclusive. Both mercury and copper dust were used to remove
"sulfur" interferences. Although it appears likely that heptachlor does not exist as
such in water or sediment due t o its rapid conversion to l-hydroxychloride by simple
chemical h y d r o l y ~ i s ,the
~ ~ question still remains whether the virtual nonexistence of
heptachlor in these samples could also be due to the mercury or copper dust treatment.
H. Effects of CO-Extractives
Generally, there are two types of undesirable effects caused by sample CO-extractives.
One is caused by those CO-extractivesthat exhibit similar analytical behavior as the
analytes in question. These CO-extractivesare referred to as "artifacts". The other
effect is caused by those CO-extractivesthat do not exhibit similar analytical behavior
to the analytes in question, but rather interfere with the analyses. We shall briefly
examine both types of CO-extractivesand their effect on analysis.
Volume 1 71
l. Artifacts
Artifacts are CO-extractivesthat exhibit similar or identical behavior as the analyte
in question causing wrong analytical interpretation. To state the presence of a contam-
inant or pollutant when in fact it is absent could result serious political, financial, and
legal implications depending on the purpose of the analytical data. Confirmation of
identity such as by chemical derivatization (see Chapter 3) or by G U M S technique is
therefore a basic quality control activity in any analytical laboratory. An artifact can
have a similar or identical retention time, R, value, p-value, as the analyte to be iden-
tified. For example, Lee et have reported an artifact in reagents having similar
GLC-EC characteristics as BHC or aldrin. Artifacts from certain plastics that have
similar GLC retention times and other analytical characteristics are well known. For
example, as early as 1964, Burke and Guiffridaso observed that a petroleum ether ex-
tract of a polyethylene (wash) bottle showed a large response by both electron capture
and microcoulometric GLC. In the same study, microcoulometric GLC also showed
that the material was halogenated and that retention times of some of the peaks pro-
duced by reagent blanks were very similar to those of certain pesticides. A more dra-
matic example is perhaps the observation of two artifacts in certain petroleum ether
as reported by Mestres et al. in 1966."l They have the retention times as o,p'- and p,p'-
DDE on a DC-200 and a QF-1 column using ECD and microcoulometric detector in
the first case. Furthermore, the p-values of petroleum ether/acetonitrile partitioning
between p , p D D E and one of the mentioned artifacts are very similar (0.58 and 0.62,
respectively). An artifact which behaved like aldrin on a DC-200 column or QF-l/DC-
200 column was also found8' in reagents and soil samples. It was identified as elemental
sulfur, which was also present in many sediment samples as discussed earlier. The
problem of sulfur interference particularly with aldrin was also discussed by Osadchuk
and W a n l e ~ s , "who
~ mentioned that confirmation of aldrin (in food samples) on TLC
at times was difficult because of the much lower sensitivity of this technique. These
authors found that neither FlorisilB nor charcoal, both widely used for the cleanup
of extracts, was effective in the complete removal of the above interfering substances.
Therefore, confirmation by chemical derivatization was r e ~ o m m e n d e d Artifacts .~~ of
dieldrin and DDT from rubber (automobile tire), gasoline, engine oil, etc. have also
have been noted.84 Since these materials are common garbage in polluted water sys-
tems, the analyst may wish to bear in mind these interferences and artifacts when
analyzing water and sediment samples. Two artifacts of dieldrin were foundss in green
plant materials such as alfalfa, wheat, corn, kale, clover, and grass. These artifacts
were not easily detected under normal GLC conditions and they resisted removal by
the common cleanup procedures for plant material."' The authors further commented
that
. . . because of resolution and retention time considerations and to minimize liquid phase bleed-off, EC-
GLC analyses of organochlorine pesticide residues are most often conducted at column temperatures o f 190
to 210°C. At these temperatures the dieldrin artifacts cannot be adequately separated from the insecticide.
Chromatography o n a nonpolar liquid phase such as DC-200 produces a "dieldrin" peak that is slightly
skewed with some base-line broadening, indicating a multicomponent peak. Increasing the liquid phase
polarity decreases the resolution. The more polar QF-l and DEGS liquid phases give a completely symrnetr-
ical "dieldrin" peak with no base-line broadening, s o that the presence of the artifacts is completely dis-
guised. Multicolumn analysis at normal column temperatures may, therefore, lead to false conclusions about
the presence of dieldrin in the test sample.
These authors illustrate an important warning in the application of the multicolumn

technique for the purpose of confirming pesticide identity.
2. Interferences
While artifacts behave like the analytes in question and hence cause misidentifica-
tion, interferences d o not necessarily behave like the analytes but nevertheless affect
the analyses. In severe cases, interferneces can cause complete failure of the analyses.
Some examples of interferences are the notorious broad solvent fronts observed
from the use of some solvents, reagents, and certain sample extracts during GLC/ECD
analyses. Rubber products such as rubber tubings are notorious for these interfering
materials. Anhydrous sodium sulfate has been knowns2.86-88 to contain interfering ma-
terials which confuse GLC interpretation. Distilled is another common
source of interference. For trace organic analysis at sub ppt levels it is quite often a
problem to obtain sufficiently "pure" water for spiking experiments. Several commer-
cial distillation units with all glass and ion-exchange column that we have tested do
not always produce sufficiently pure water for this low level investigation. In this case
the water is further treated with a XAD-2 column (see Volume 11, Chapter l ) or with
a solvent extraction technique to remove the last traces of interferences.
As mentioned previously, some impurities of solvents do not cause GLC interfer-
ences but rather affect the analytical process such as to cause overlapping of fractions
in column chromatographic cleanup.
For TLC the presence of starchy or oily CO-extractivescan modify the R, values of
the analyte in question. Unfortunately, there are few guidelines or warnings for the
analyst to suspect this happening except by experience and "analytical intuition". An
early personal experience (in 1965) by one of us was the analysis of potato extracts for
carbaryl by TLC. Due to the excessive starch present, the R, values of carbaryl in the
sample extracts were sufficiently different from those of the standards run side by side
on the TLC plate to cause a false conclusion on its absence. Fortunately, the color of
the spot in the sample was very intense and similar to that from carbaryl so that further
tests (GLC, chemical derivatization) were carried out to confirm the presence of car-
baryl even though TLC indicated its absence.
VII. THIN-LAYER CHROMATOGRAPHY (TLC)
A. Introduction
TLC has now replaced the less sensitive and efficient paper chromatography. The
simplicity, economy, speed, versatility, and better spot definition of TLC are some
factors which constitute its popularity in separation and analysis. In pesticide residue
analysis, TLC has been and is still used for the qualitation and quantitation of residues.
Its application to the analysis of organophosphorus pesticides (o.p.s), carbamates, and
ureas has been discussed in the appropriate chapters. The use of TLC for organochlo-
rine pesticides is particularly limited mainly because of its insufficient sensitivity as
compared to the generally used ECD for analysis. In general, TLC is mainly used as a
supplementary rather than the main cleanup technique. This is because often more
vigorous cleanup is required for TLC than for GLC if streaked zones are to be avoided
or minimized. Lipids and starchy materials are some sample CO-extractiveswhich must
be substantially reduced to give good resolution in TLC. On the other hand, in many
cases presence of these materials can be tolerated in GLC analysis even using ECD.
Consequently, application of TLC as a cleanup technique is limited because sample
extracts which are adequately cleaned up for GC analysis often require additional
cleanup before TLC determination can be performed. In situ quantitation of TLC
plates is possible by using a densitometer to scan sample and standard spots. The de-
gree of reflectance or transmission of each spot, depending on the operation mode
used, is translated into the correspondence peak which is proportional to concentra-
tion. Calculation of concentration is by comparision with a known standard in the
same manner as for GC peaks. For general quantitation, TLC with densitometer could
be less convenient than GLC. Among other factors, the need of vigorous cleanup for
sample extracts before TLC determination is a major disadvantage. For some com-
pounds, such as 0.c.s and PCBs, sensitivity is another factor. It also requires that
Volume I 73
instrumentation and the TLC operation must be rigidly controlled for precise and ac-
curate results. For examples, precise spotting, uniform TLC layers, suitable R,, uni-
form application of detection reagents, and amount and type of sample CO-extractives
can affect the reliability of results. Also, the resolution of TLC for multi-residue anal-
ysis is inferior to that from the GLC column. Consequently TLC is not oftenly used
as a quantitation technique for multi-residue analysis.
Following initial screening and quantitation by GLC, TLC can be used as a semicon-
firmatory technique to provide further substantiation to pesticide residue identity. In
this application, TLC is used in conjunction with GLC or less frequently, spectropho-
tometry. The technique of TLC-GLC will be discussed later.
In this section, the basic principles and techniques of conventional TLC for pesti-
cide-residue analysis is discussed. A modified procedure developed in 1966 by Chau is
also discussed.
B. Adsorbents
The versatility of TLC is due to the variety of adsorbents available to coat on a solid
support such as a glass plate. Silica gel is traditionally by far the most widely used
adsorbent for pesticide residue analyses. Silica gel G is a popular form of finely POW-
dered silica gel with calcium sulfate as binder whereas silica gel H uses silicon dioxide/
aluminum oxide as binder. Finely powdered alumina with calcium sulfate as binder is
also commonly used in pesticide residue work. Some other TLC adsorbents are Flor-
isilO, Kieselguhr G, magnesium silicate, cellulose powder, and miscellaneous inor-
ganic adsorbents. In some applications, mixed adsorbents or an adsorbent mixed with
chemical are other approaches to enhance separation or improve resolution of spots.
An adsorbent impregnated with silver nitrate is perhaps the most common example
in certain pesticide residue application. This application is said to be advantageous for
improving separation of aromatic from nonaromatic compounds. Recently, commer-
cially available reversed-phase TLC plates put some new dimensions in TLC method-
ology by increasing sample loading capacity, improving resolution and sensitivity com-
pared to conventional TLC. Like the adsorbent used in high-pressure liquid
chromatography, reversed-phase TLC plates use a manufactured process by derivatiz-
ing the surface silanol groups (Si-0-H) with octadecyl (C,s) groups such as by using
octadecylsilane. This creates a hydrocarbon surface to the solutes. The principal sepa-
ration mechanism of reversed-phase TLC or a reversed-phase column in HPLC is the
Van der Waals' attraction between the hydrocarbon moiety of the analyte and the
octadecyl groups on the surface of the adsorbent. More details on reversed-phase TLC
will be discussed later.
C. Coating of T L C Plates
Commercially coated TLC plates offering several varieties of adsorbents are readily
available. Due to the uniform thickness of the coatings, they have better reproducibil-
ity than home made plates. However, for many applications use of the home made
plates according to or similar to Stahl's procedureg0 is satisfactory and can be more
economical. Also, it is desirable on certain occasions to prepare one's own plates so
that TLC plates of a variety of mixed adsorbents can be readily made.
Use TLC grade alumina or silica gel containing gypsum (calcium sulfate) inorganic
binder to prepare TLC plates. The inertness of gypsum allows all destructive charring
and ashing techniques, since it is unreactive to chemical and biochemical chromogenic
sprays used in pesticide residue work. However, since the slurry prepared from these
materials becomes too viscous to spread in less than 5 min, all equipment and materials
should be ready for the coating operation before the slurry is prepared. Following is
the procedure for coating TLC plates:
1. Clean the glass plates (200 mm X 200 mm, or other chosen size) with soap and
water; rinse with acetone so that they are free from oily materials and other
contaminants (oily glass plates give uneven thin-layer plates). Dry the glass plates
in air at room temperature or in a warm oven. Arrange 5 clean plates in a
spreader with a smaller plate on each end, making sure all the plates are of uni-
form thickness and width.
2. In a glass-joint conical flask (250 ml), add 30 g silica gel followed by 50 m l
distilled water (free of chloride). Alternatively, silica gel can be ground with water
in a porcelain mortar. Stopper the flask and shake evenly for 30 to 60 sec; gentle
tumbling works fine. Vigorous shaking produces bubbles and results in uneven
plates. Immediately after shaking, pour the slurry into the trough of a TLC coat-
ing applicator sitting on the glass plates which are arranged on a spreader. Apply
a layer 0.25 mm thick on the 5 glass plates with rapid but even movement. (Air
bubbles or lumps in the slurry must be avoided if good TLC plates are to be
prepared.)
3. Allow the plates to dry at ambient temperature for 10 to 20 min and transfer the
coated plates to a rack. Activate the plates in a vertical position for 1 hr at 110°C.
Immediately store plates in a desiccator. (Before use, a plate can be activated
again for a few minutes or used as is. To prevent inconsistent results the same
drying and storage procedures should be followed each time a new set of coated
plates are made. It should also be noted that during hot humid weather results
vary from plate to plate when exposed to humidities of greater than 50%. For
this reason, the plates should be used in a dehumidified room if possible and
activated for a fixed period [e.g., 30 min] each time they are used in hot humid
weather. In any case, unnecessarily long exposure of plates to the atmosphere
must be avoided.)
Note - The usefulness of the common practice to use some desiccants, such
as indicating silica gel in a desiccator to store prepared TLC plates, has been
questioned. The activated TLC plates have greater moisture adsorption capability
than the less efficient indicating silica gel so that this desiccant is kept dry by the
TLC plates and not vice-versa as intended. On the other hand, there is no point
in using more effective desiccants such as P,O, because moisture is almost im-
mediately adsorbed when the TLC plates are exposed to lab atmosphere. There-
fore, it has been suggested that after activation, the plate be equilibrated with
the desired humidity.
D. Spotting
Evaporate a suitable aliquot of clean-up sample extract to a very small volume
(about 100 PP). Using a spotting template as a guide, apply the sample extract on one
side and the standard on the other by means of a micropipette or a microsyringe (25
p1 or 10 p l is convenient for most applications) at a distance of 10 cm from each spot
and 25 mm from the bottom edge of the plate. Although as many as 18 spots can be
put on a single 200-cm X 200-cm plate for standard solutions, a maximum of 10 to 12
spots is recommended to avoid overlapping and slanting in some samples.
E. Development and Visualization

l . Preparation
Preparation of development tank: For 200-cm X 200-cm TLC plates use a rectangular
tank containing 150 m1 to 200 m1 of developing solvent or solvent mixtures, (see Table
7 on choice of developing solvents). Line both sides of the tank with two pieces of
filter paper, arranging the tank so that the filter papers are well soaked with solvent.
After the papers are moistened, press the papers against the side of the tank. Cover
Volume I
Table 7
ELUOTROPIC SERIES OF
SOLVENTS
mHexane Lowest polarity

Heptane
Cychlohexane
Carbon tetrachloride
Toluene
Benzene
Chloroform
Tetrahydrofuran Intermediate
Ethyl ether
Ethyl acetate
Pyridine
Acetone
mPropanol
Ethanol
Methanol
Water
Acetic acid Highest
Note: Several authors have slightly different

versions of this series; however, this
variation does not appreciably affect
the results on the choice of solvents.
the development tank tightly (silicone grease applied on tip of the cover) and let stand
at room temperature for a few minutes to allow saturation of the solvent vapor within
the chamber. These steps are necessary to avoid the "edge effect" that would result
from inadequate chamber solvent ~ a t u r a t i o n . ~
This
' phenomenon is indicated by sub-
stances near the edge of the plate migrating higher than the same substances in the
center.
2. Developing Solvents
The choice of developing solvent or mixture of solvents depends on the polarity of
compounds. A general rule is that the more polar the compounds, the more polar the
development solvent(s) used. The polarity of solvents is given in the eluotropic series
of solvents in Table 7.
3. Visualization
After development (i.e., the solvent moves up to a previously marked height, about
15 cm from top of plate), transfer the developed plate to a fumehood to evaporate the
solvent (approximately 5 min). In the fumehood, spray the whole plate evenly 2 or 3
times with an appropriate chromogenic agent.
For O.C.pesticides, a chemical chromogenic spraying reagent followed by UV irra-
diation is used to visualize the spots on the TLC plates (see chapter on o.c.).
For 0.p. and carbamate pesticides both chemical or biochemical (enzyme) spraying
agents have been used. The enzyme inhibition technique is the most sensitive (see chap-
ters on carbamate and 0.p.s).
F. TLC-GLC Method
Instead of using chromogenic spray reagent, with or without UV irradiation for
visualization of the sample spots, only the standards are exposed to this treatment for
visualization. The corresponding areas on the remainder of the plate are respectively
removed and extracted with an appropriate organic solvent. After filtration and con-
centration, if necessary, the extracts can be examined by GLC for identification. If
suitable, IR, UV spectroscopy, MS, colorimetric technique can also be used.
The procedure of TLC-GLC is described as follows:
1. Spot aliquots of sample extracts on the center portion of a TLC plate and stand-
ard solution (single o r mixed) on both sides of the plate. Develop the plate as
usual. Cover the sample (center) portion with a piece of cardboard, taking care
not to scratch the coating. Spray with an appropriate reagent (see respective chap-
ter for choice). For O.C. compounds, irradiate the covered sample portion with
UV lights until dark spots appear on the standard portion. For other pesticides,
no UV irradiation is generally necessary.
2. In the sample area, draw a line 1/2 cm above and below each of the correspond-
ing standard spots. It should be noted that excess CO-extractivein the sample may
move the residue area outside the corresponding standard region. Use suction
(zone extractor) t o scrape the area, and extract with 1 m! hexane (for 0.c.s) or
other appropriate solvent such as benzene or acetone for more polar residues.
After the adsorbent settles down, the supernatant liquid is examined by GLC. If
concentration is required, filtration and washing of residues is necessary before
evaporation of extracting solvent.
The method used here is not for cleanup, but for confirmation purposes. Instead of
spraying and irradiating the whole plate for visualization, GLC is used to "visualize"
an area which corresponds t o a particular standard. Visualization by chromogenic
spray agents is often useless for the analysis of 0.c.s residue in environmental samples
as they are usually below the detection limit of TLC. Some fish and wildlife lipid
samples are exceptions. However, "visualization" by GLC works quite well.
For water and most sediment samples, the CO-extractivesare minimal and d o not
affect the R, value of the residue as compared to the corresponding standard.
For samples that contain a complicated mixture of pesticides of different polarity,
two plates are generally used, one with low polarity solvents (e.g., 0.5 m1 acetone in
200 m! hexane), the other with a more polar solvent (e.g., 1.0 to 2. 0 m! of acetone
in 200 m1 hexane). A better separation of pesticides results. For two pesticides with
close R, values, the area scraped and extracted with hexane will contain both pesti-
cides, which usually does not hamper the interpretation so long as they have different
GLC retention times.
G . Discussion
TLC is widely used for confirmation of identity after preliminary GLC analysis.
Enzyme inhibition technique (such as using liver extract as spraying agent) for 0.p.s
and some carbamate can approach and sometime even exceed the sensitivities of GLC
with flame photometric detector. For o.c.s, applicability of TLC is greatly hampered
by its relatively low sensitivity as compared to GLC-ECD; however, this difficulty is
resolved with the above-discribed technique of pairing GLC with TLC. Although TLC
in general and TLC-GLC in particular are useful tools for confirmation, one should
still be careful in interpreting results based on these techniques. As discussed in Chap-
ter 3 on chemical confirmatory tests, many compounds are known to have very similar
or identical R, values, e.g., sulfur and aldrin, 2-chlorochlordene and heptachlor, DDT
isomers and metabolites.
Developing solvents for TLC varies from worker to worker, due to conditions vary-
ing from one laboratory to another. One must find the right solvents for the developing
of TLC plates, by trial and error.
Volume I 77
Several important points are emphasized here:
1. Due to the high adsorptive capacity of silica gel plates, considerable care must
be exercised in handling and storage to avoid adsorption of vapors of interfering
substances.
2. The activity of prepared TLC plates changes with the length of storage and
amount of handling, even if kept well in a desiccator. The solvent system for
these plates found applicable for a certain compound may be unsatisfactory after
a few months for the same compound. Therefore, after long storage it is advisa-
ble t o reactivate the plates before use and check the suitability of the solvent
system on standards before doing samples which do not have enough material
for repetition.
3. Developing solvent mixtures should not be reused after developing TLC plates
unless they contain one kind of solvent. This is particularly important in a solvent
mixture containing a small percentage of one solvent in another.
4. Values of pesticides change in the presence of some CO-extractives.For example,
starch as a CO-extractive in certain vegetation modified the R, values of some
pesticides. For this reason, the extracts of samples with which the analyst is un-
familiar should have pesticide standards added to them and applied on a TLC
plate. A comparison should then be made with a standard in order to observe
any changes in R, values of pesticides due to CO-extractives.
H. Other Forms of T L C
The above discussion so far focuses on the "normal" TLC operation, using classical
TLC technique. There are various forms of TLC operations such as hot plate TLC
prepared by warming a chromatographic plate during the TLC process to cause the
solvent to evaporate in order to achieve better resolution and sen~itivity,~' glass rod
TLC using a glass rod as solid support rather than a plate,93 two-dimensional TLC,
multi-dimensional TLC, wedge-shaped development, and so on.
In pesticide residue analysis, besides the "normal" TLC technique, perhaps the next
commonly used technique is the two-dimensional TLC to achieve better resolution of
a complicated mixture. By this technique, a second development is applied, perpendic-
ular to the first direction from the first development. In other words, after the first
development the plate is inverted 90" for the second development. The same or a dif-
ferent solvent can be used for each development. The advantage of this technique is
enhancement of resolution and minimization of interference from artifacts. However,
it is time consuming and not easy to reproduce. For labile compounds, there is a greater
chance of degradation due to increased exposure to atmosphere.
1. Reversed-Phase TLC (R P-TLC)

The conventional TLC conditions use a polar stationary phase and a nonpolar sol-
vent, whereas the RP-TLC uses a nonpolar phase and a polar solvent such as methanol
and water. RP-TLC technology is an extension of the reversed-phase column technol-
ogy of high pressure liquid chromatography (HPLC).
In RP-TLC, as in RP-HPLC, the polar compounds are eluted or move faster (larger
R, values) than the nonpolar compounds by a less polar solvent. For example, in the
commonly used organic solvent-water mixture for RP-TLC, the more nonpolar the
compound the higher the proportion of organic solvent used. As the polarity of com-
pounds to be separated increases, the water content of the developing solvent also
increases. The advantages of RP-TLC in comparison to conventional TLC are in-
creased resolution, higher sample loading capacity, and efficiency. Most important of
all, RP-TLC does not correlate with the conventional column chromatography com-
monly used for sample cleanup thus providing an additional dimension on confirma-
tion of residue identity as yet, it is a relatively new area and more application of RP-
TLC on residue analysis remains to be developed.
2. High-Performance TLC (HPTLC)

HPTLC is a relatively new technique of increasing importance and popularity. By
incorporating special types of TLC plates, new spotting techniques, and in situ densi-
tometry, HPTLC separates mixtures in nanongram and picogram quantities at high
speed yet with much improved resolution over classical TLC. The extraordinary per-
formance of HPTLC is achieved by using high purity adsorbents of smaller particle
size (5 to 10 pm), narrow particle size distribution, and uniform layers. In order for
the above advantages of HPTLC to be fully realized, volumes less than 1 p l must be
spotted with spot diameters of less than 1 mm at the origin. Plates are developed in a
small glass tank and the resultant chromatograms have tight (2 to 3-mm diameter) and
well-separated spots. Commercial HPTLC plates coated with silica gel for normal
phase separation or chemically bonded C 2 , C S ,and C,s silica gel for reversed phase
separation are both available. Because of the loading limitation, HPTLC is not yet
widely used in pesticide residue work. Yet, with a similar adsorbent, the data derived
from HPTLC and HPLC are often highly correlated; thus the former can be used in
early method development work such as the optimization of solvent systems before
using the more sophisticated and tedious HPLC system in routine analysis.
REFERENCES
1. Jones, L. R. and Riddick, J. A., Separation of organic insecticides from plant and animal tissues,
Anal. Chem., 24, 569, 1952.
2. Thornburg, W. W., Preparation and extraction of samples prior to pesticide residue analysis, J.
Assoc. O f f .Agric. Chem.,48, 1023, 1965.
3. Morley, H. V., Adsorbents and their application to column cleanup of pesticide residues, Res. Rev.,
16, 1, 1966.
4. Brockmann, H. and Schodder, H . , Alurnina oxide with buffered adsorptive properties for purposes
o f chromatographic adsorption, Ber. dtsch. chem. Ges., 74,73, 1941.
5. Kadoum, A. M., Activation and standardization of purified silica gel for column chromatographic
cleanup of pesticide residue extracts, Bull. Environ. Cont. Toxicol., 4, 120, 1969.
6. Kawano, Y., Bevenue, A., Beckman, H., and Erro, F., Studies on the effect of sulfuric-fuming nitric
acid treatment on the analytical characteristics of toxaphene, J. Assoc. O f f . Anal. Chem., 52, 168,
1969.
7 . Klein, A. K. and Link, J. D., Elimination of interferences in the determination of toxaphene residues,
J . Assoc. O f f .Anal. Chem., 53, 524, 1970.
8. Risebrough, R. W., Reiche, P., and Olcott, H. S., Current progress in the determination of the
polychlorinated biphenyls, Bull. Environ. Cont. Toxicol.,4, 192, 1969.
9. Widmark, G . , Possible interference by chlorinated biphenyls in IUPAC Commission on methods of
pesticide residue analysis, J. Assoc. O f f .Anal. Chem., 50, 1069, 1967.
10. Jan, J. and Malnersic, S., Determination of PCB and P C T residues in fish tissue by acid hydrolysis
and destructive cleanup of the extract, Bull. Environ. Cont. Toxicol., 19, 772, 1978.
11. Holdrinet, M. V. H., Ontario Ministry of Agriculture and Food, Guelph, Ontario, private comrnu-
nication to A.S.Y. Chau, 1974.
12. Norstrom, R. J., Won, H. T., Holdrinet, M. V. H., Calway, P. G . , and Naftel, C. D. Gas-liquid
chromatographic determination of mirex and photomirex in the presence of polychlorinated biphen-
yls: interlaboratory study, J. Assoc. O f f .Anal. Chem., 63, 37, 1980.
13. Trotter, W. J . , Removing the interference of DDT and its analogs in the analysis for residues of
polychlorinated biphenyls, J. Assoc. O f f .Anal. Chem., 57,461, 1974.
14. Miles, R. J. W., Conversion of DDT and its metabolites to dichlorobenzophenones for analysis in
the presence of polychlorinated biphenyls, J. Assoc. Off. Anal. Chem., 55, 1039, 1972.
15. Gothe, R., Oxidation with tetrabutylammonium permanganate for quantitation of DDT residues in
GLC determination of chlorinated hydrocarbons, Bull. Environ. Cont. Toxicol., l l , 451, 1974.
16. Chau, A. S. Y. and Luft, B., Effect on the recoveries of 10 0.c.s at ppb levels during acid-base
partitioning, unpublished report, Water Quality Branch, Inland Waters Directorate, Environment
Canada, Burlington, Ontario, 1973.
17. Coburn, J., Ripley, B. D.,and Chau, A. S. Y., Analysis of pesticide residues by chemical derivatiza-
tion, 11. N-methylcarbamates in natural water and soils, J. Assoc. Off. Anal. Chem., 59, 188, 1976.
18. McLeod, H. A. and Wales, R. J., A low temperature cleanup procedure for pesticides and their
metabolites in biological samples, J. Agric. Food. Chem., 20,624, 1972.
19. Tindle, R. C. and Stalling, D. L., Automated gel permeation cleanup of extracts for pesticide residue
analysis. I. General system, Anal. Chem.,44, 1768, 1972.
20. Stalling, D. L., Tindle, R. C., and Johnson, J . L., Cleanup of pesticide and polychlorinated biphenyl
residues i fish extracts by gel permeation chromatography, J. Assoc. Off. Anal. Chem,. 55, 32, 1972.
21. Griffith, K. R. and Craun, J. C., Gel permeation chromatographic system: an evaluation, J. Assoc.
Off. Anal. Chem.,57, 168, 1974.
22. Johnson, L. D., private communication to A. S. Y. Chau, 1974.
23. Suggested procedure for nonionic chlorinated pesticides in the lipid of poultry, swine, beef, soybeans
and corn prepared for analysis by GPC, Analytical Biochemistry Laboratories, Inc., Columbia, MO.
24. Sherma, J., Manual of Analytical Quality Control for Pesticides and Related Compounds in Human
and Environmental Samples, U.S. Environmental Protection Agency, Research Triangle Park, N.C.,
1979,90.
25. McReynolds, W. O . , Characterization of some liquid phases, J. Chromatogr. Sci., 8,685, 1970.
26. Supina, W. R., The Packed Column in Gas Chromatography, Supelco Inc., Bellefonte, 1974.
27. vanDeWeil, H. J. and Tommassen, P., Effect of oxygen on electron-capture detection, J . Chrorna-
togr.,7l, 1, 1972.
28. Perretta, A. T., Carrier purity in gas chromatography, Am. Lab., 35, May 1976.
29. Walker, E. A. and Palframan, J. F., Techniques in gas chromatography. 11. Developments in the
van Deemter rate theory of column performance. A review, Analyst, 94,609, 1969.
30. Grant, D. W., Column development and technology in gas chromatography, J. Chromatogr., 122,
107, 1976.
3 1. David, D. J, Gas Chromatographic Detectors, Interscience, New York, 1974.
32. Hall, R. C., The nitrogen detector in gas chromatography, C R C Crit. Rev. Anal. Chem., 7, 323,
1978.
33. Hewlett-Packard Operating Manual for Gas Chromatograph Series 5700A, Avondale, Pa., December
1972, 3.
34. The Pulsed Electron Capture Detector (Applications and Trouble-shooting Manual), Hewlett-Pack-
ard, Avondale, Pa.
35. Lovelock, J . E., Electron Absorption detectors and technique for use in quantitative and qualitative
analysis by gas chromatography, Anal. Chem., 35,474, 1963.
36. Maggs, R. J., Joynes, P . L., Davies, A. J., and Lovelock, J . E., The electron capture detector - a
new mode of operation, Anal. Chern.,43, 1966, 1971.
37. Aue, W. A. and Kapila, S., The electron capture detector - controversies, comments and chroma-
tograms, J . Chrornatogr. Sci., l l , 255, 1973.
38. Karmen, A. and Giuffrida, L., Enhancement of the response of the hydrogen FID to compounds
containing halogens and phosphorus, Nature (London), 201, 1204, 1964.
39. Kolb, B. and Bischoff, J., A new design of a thermionic nitrogen and phosphorus detector for GC,
J. Chrornatogr. Sci., 12,625, 1974.
40. Bente, H. B., Applications and Operation of the Nitrogen-Phosphorus Detector, Application Note
AN 228-4, Hewlett-Packard, Avondale, Pa., 1977.
41. Bowman, M. C. and Beroza, M., Gas chromatographic detector for simultaneous sensing of phos-
phorus- and sulfur-containing compounds by flame photometry, Anal. Chern., 40, 1448, 1968.
42. Dolan, J. W. and Hall, R. C., Enhancement of the sensitivity and selectivity of the Coulson electro-
lytic conductivity detector to chlorinated hydrocarbon pesticides, Anal. Chern, 45, 2198, 1973.
43. Elgar, K. E., The identification of pesticides at residue concentrations, in Pesticides Identification at
the Residue Level, Advances in Chemistry Series, 104, American Chemical Society, Washington,
D.C., 1971, 151.
44. Thompson, J. F., Walker, A. C . , and Moseman, R. F., Evaluation of eight gas chromatographic
columns for chlorinated pesticides, J. Assoc. Off. Anal. Chem., 52, 1263, 1969.
45. Robinson, J., Residues of organochlorine insecticides in dead birds in the United Kingdom, Chem.
Ind.,p. 1974, 1967.
46. Reynolds, L. M., PCBs and their interference with pesticide residue analysis, presented to the Eastern
Canada Seminar on Pesticide Residue Analysis, Guelph, Ontario, 1968.
47. Beroza, M. and Bowman, M. C., Extractions of insecticides for cleanup and identification, J. Assoc.
Off. Agric. Chem., 48,358, 1965.
48. Bowman, M. C. and Beroza, M., Extraction p-values of pesticides and related compounds in six
binary solvent systems, J. Assoc. Off. Agric. Chem., 48,943,1965.
49. Beroza, M. and Bowman, M. C., Identification of pesticides at nanogram level by extraction p-
values, Anal. Chem.,37, 291, 1965.
50. Beroza, M. and Bowman, M. C., Apparatus and procedures for rapid extraction and identification
of pesticides by single and multiple distribution in binary solvent systems, Anal. Chem., 38, 837,
1966.
51. Beroza, M., Inscoe, M. M., and Bowman, M. C., Distribution of pesticides in immiscible binary
solvent systems for cleanup and identification and its application in the extraction of pesticides in
milk, Res. Rev.,30, 1, 1969.
52. Giang, P. A., Library of infrared spectra of important pesticides, in Analytical Methods for Pesti-
cides and Plant Growth Regulators, Vol. 9, Zweig, G. and Sherma, J., Eds., Academic Press, New
York, 1977,153.
53. Grob, R. L., Ed., Chromatographic Analysis of the Environment, Marcel Dekker, New York, 1975.
54. Velde, G. V. and Ryan, J. F., Gas chromatography-mass spectrometry as applied to pesticide analy-
sis, J. Chromatogr. Sci., 13,322, 1975.
55. Pagel, J. E., Microbiology Quality Assurrance Programs, Data Quality Report Series, Ontario Min-
istry of the Environment, 1976.
56. Handbook for Analytical Quality Control in Water and Wastewater Laboratories, Office of Research
and Development, U.S. Environmental Protection Agency, Cincinnati, Ohio, 1979.
57. Anon., Quality control procedures and objectives, Data Quality Report Series, Ontario Ministry of
the Environment, 1976.
58. Bretthauer, E. W.,Morgan, G. B., and Jaquish, R. E., Standard reference materials and environ-
mental monitoring, in Standard Reference Materials and Meaningful Measurements, Special Publ.
408,National Bureau of Standards, Washington, D.C., 1975.
59. McCully, K. A., Analysis of multiple pesticide residues - chemical identity and confirmation of
results, WorldRev. Pest. Cont., 8,59,1969.
60. Burke, J. A., Mills, P. A., and Bostwick, D. C., Experiments with evaporation of solutions of chlor-
inated pesticides, J. Assoc. Off. Anal. Chem.,49,999,1966.
61. Chiba, M. and Morley, H. V., Studies of losses of pesticides during sample preparation, J. Assoc.
Off. Anal. Chem., 51, 55, 1968.
62. Chau, A. S. Y., unpublished results, 1970-1972.
63. Ripley, B. D.,Wilkinson, R. J., and Chau, A. S. Y., Multiresidue analysis of fourteen organophos-
phorus pesticides in natural waters, J . Assoc. Off. Anal. Chem., 57, 1033,1974.
64. Coburn, J. A., Ripley, B. D., and Chau, A. S Y., Analysis of pesticide residues by chemical derivi-
tization. 11. N-methyl carbamates in natural water and soils, 3. Assoc. Off. Anal. Chem., 58, 1294,
1976.
65. Agemian, H. and Chau, A. S. Y., Analysis of pesticide residues by chemical derivatization. V. Mul-
tiresidue analysis of eight phenoxyalkanoic acid herbicides in natural waters, 3. Assoc. Off. Anal.
Chem., 60, 1070,1977.
66. Agemian, H. and Chau, A. S. Y., Determination of pesticides by derivative formation. 1V. A sensi-
tive gas-chromatographic method for the determination of MCPA and MCPB herbicides after ester-
ification with I-bromoethyl-2,3,4,5,6-pentafluorobenzene, Analyst, 101,732,1976.
67. U.S. Pesticide Analytical Manual, Vol. 1, Food and Drug Administration, Washington, D.C., 1972.
68. Analytical Methods Manual, Inland Waters Directorate, Water Quality Branch, Environment Can-
ada, Ottawa, 1979.
69. "Moisture kills columns", Bull. 739,Supelco Inc., Bellefonte, Pa.,
70. Yip, G., Determination of herbicides in oils, J. Assoc. Off. Agric. Chem.,47, 1 1 16,1964.
71. Chau, A. S.Y., unpublished results, 1973.
72. Goerlitz, D. F. and Lamar, W. L., Determination of phenoxy acid herbicides in water by electron-
capture and microcoulometric gas chromatography, Geologic1 Survey Water-Supply paper 1817-C,
U.S. Government Printing Office, Washington, D.C., 1967.
73. Zitko, V., Effects of pesticide-grade hexames on the silicic aci chromatography of polychlorinated
biphenyls and organochlorine pesticides, J. Chromatogr., 59,444,1971.
74. Bevenue, A.,KeUey, T. W., and Hylin, J. W., Problems in water analysis for pesticide residues, J.
Chromatogr., 54,71,1971.
75. Williams, I. H., Photochemical oxidation of hexane isomers to compounds sensitive to electron-
capture detection, J. Chromatogr. Sci., l l, 593,1973.
76. Thompson, J. F., Ed., Analysis of Pesticide Residues in Human and Environmental Samples, U.S.
Environmental Protection Agency, Research Triangle Park, N.C., 1976.
77. Goerlitz, D. F. and Law, L. M., Note on removal of sulfur interferences from sediment extracts for
pesticide analysis, Bull. Environ. Cont. Toxicol.,6, 9, 1971.
78. Miles, J. R. W., Tu, C. M., and Harris, C. R., Metabolism of heptachlor and its degradation prod-
ucts by soil microorganisms, J . Econ. Entomol., 62, 1334, 1969.
78a. Aspila, K. I., Carron, J. M., and Chau, A. S. Y., Interlaboratory quality control study of the analysis
of water for pesticides, J. Assoc. O f f .Anal. Chem., 1097, 1977.
79. Lee, D. F., Britton, J., Jeffcoat, B., Mitchell, R. F., Interference with the determination of aldrin
and benzene hexachloride residues by gas chromatography, Nature (London), 21 1, 521, 1966.
80. Burke, J. and Giuffrida, L., Investigations of electron capture gas chromatography for the analysis
of multiple chlorinated pesticide residues in vegetables, J. Assoc. O f f .Agric. Chem., 47, 326, 1964.
81. Mestres, R., Barthes, F., and Priu, M. M., Residue de pesticides. Sur bne cause d'erreur dans la
recherche des insecticides organochlores, Soc. Pharrnacie Montpellier, 26, 93, 1966.
82. Pearson, J. R., Aldrich, F. D., and Stone, A. W., ldentification of the aldrin artifact, J. Agric.
FoodChern., 15,938, 1967.
83. Osadchuck, M. and Wanless, E. B., ldentification and quantitative estimation of aldrin residues in
the presence of interfering materials in samples of plant and animal origin, J. Assoc. O f f . Anal.
Chern.,SI, 1264, 1968.
84. Denbert, K. H., Sources of compounds interfering with the analysis of extracts for dieldrin and DDT
by GLC, Bull. Environ. Cont. Toxicol., 5, 379, 1970.
85. Glotfelty, D. E. and Caro, J. H., Artifacts of dieldrin in gas chromatographic analysis of green plant
material, Anal. Chern.,42, 282, 1970.
86. Lamar, W. L., Goerlitz, D. F., and Law, L. M., Determination of organic insecticides in water by
electron capture gas chromatography, Adv. Chern. Series, 60,207, 1966.
87. Chau, A. S. Y., Analysis of chlorinated hydrocarbon pesticides in waters and wastewaters, Depart-
ment of the Environment, Ottawa, Canada, 1972.
88. Levi, I. and Nowicki, T. W., Interfering GLC peaks from materials and chemicals in pesticide residue
analysis, Bull. Environ. Cont. Toxicol., 7, 193, 1972.
89. Bevenue, A., Ogata, J. N., Kawano, Y., and Hylin, J. W., Potential problems with the use of distilled
water in pesticide residue analysis, J. Chromatogr., 60, 45, 1971.
90. Stahl, E., Thin Layer Chromatography, 2nd ed., Springer-Verlag, New York, 1969.
91. Demole, E., Applications de la microchromatographic d'adsorption sur couches minces, J. Chro-
rnatogr., 1, 24, 1958.
92. Turina, S. and Jamnicki, V., Principles of Hot Plate Chromatography, Anal. Chern., 44, 1892, 1972.
93. Streator, J. T., Chambers for thin-layer chromatography using glass rods, Anal. Chern., 40, 671,
1968.
Chapter 3
POSITIVE IDENTIFICATION OF PESTICIDE RESIDUES BY

CHEMICAL DERIVATIZATION-GAS CHROMATOGRAPHIC
TECHNIQUE*
Alfred S . Y . Chau
TABLE OF CONTENTS
I. General Discussion of Several Confirmatory Techniques . . . . . . . . . . . . . . . . . . 84

A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
B. Various Approaches on Confirmation of Pesticide Residue Identity . . .85
1. Spectrochromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
2. Thin-Layer Chromatography (TLC) ....................... 85
3. Multi-Column GLC Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
4. p-Values ............................................... 86
5. Chemical Derivatization Techniques . . . . . . . . . . . . . . . . . . . . . . . 87
I1. General Discussion of Derivatization Techniques for 0.C.s . . . . . . . . . . . . . . . . 87

A. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
B. Solid-Matrix (SM) Derivatization Techniques . . . . . . . . . . . . . . . . . . . . . 89
C. Types of Solid Matrix Developed for Pesticide Confirmation . . . . . . . .91
I11. Organochlorine Pesticides (0.C.s) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

A. DDTGroup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
B. Endrin ...................................................... 99
1. Acid-Catalyzed Isomerization Procedures . . . . . . . . . . . . . . . . . . 99
2. CrC12 Reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
C. Dieldrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
D. Aldrin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
E. The Chlordane Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
1. Heptachlor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
2. Heptachlor Epoxide and Chlordane Isomers . . . . . . . . . . . . . . . 113
F. Miscellaneous 0.C.s ......................................... 115
1. Endosulfans (a-and P-isomers) . . . . . . . . . . . . . . . . . . . . . . . . . . 115
2. Mirex ................................................. 120
3. Kepone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .122
4. Lindane and Other BHC Isomers . . . . . . . . . . . . . . . . . . . . . . . . . 123
5. HCB(Hexach1orobenzene) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
IV . Other Pesticides ................................................... 131

A. Organophosphorus Pesticides (0.P.s). .......................... 131
1. Hydrolysis of 0.P.s and Derivatization of the Products . . . . . . 131
a. Derivatization of the P Moiety ..................... 131
b. Derivatization of the Phenolic or Thiophenolic Moiety 136
* The author gratefully acknowledges the suggestions and critical review of this material by W . W . Sans
(Canada Agriculture. London. Ontario). M . Chiba (Canada Agriculture. Vineland. Ontario). M . A .
Forbes and D . McGregor (Environment Canada. Ottawa. Ontario). and B . Ripley (Ontario Ministry of
Agriculture and Food. Guelph. Ontario) .
2. Derivatization of Intact 0 . P . s . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

a. Trifluoroacetylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
b. Methylation by NaH/CH, I/DMSO System . . . . . . . . . . 139
c. Chromous Chloride Reduction .................... 141
d. Chemical Oxidation .............................. 142
3. Conclusion ........................................... 142
B. Phenoxyalkanoic Acids ....................................... 142
C. Carbamates and Ureas ....................................... 144
D. Triazines ................................................... 149
1. General Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
2. Confirmation Procedure for the Parent Triazines . . . . . . . . . . . 150
3. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .153
.
4. Confirmation of Major Metabolites of Triazines ............ 154
5. Conclusion ........................................... 155
V. Overview and Selection Criteria of Chemical Derivatization Confirmation

Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .l .5 5
A. Sensitivity ..................................................156
B. LowReagentBackground . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156 .
C. Ability to Destroy Sample CO-Extractives ....................... 157
D. Inability to Produce Detector-Responsive Products with Sample Co-
Extractives ................................................. 157
E. Unique Retention Time of Derivative . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
F. Formation of One Single Derivative Peak ........................ 157
G. Formation of a Derivative Peak with Height Equal to or Higher than
the Parent Compound . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .158
H. Simplicity. Applicability. and Specificity ........................ 158
V1. Summary ......................................................... 159
References .............................................................. 160
I . GENERAL DISCUSSION OF SEVERAL CONFIRMATORY

TECHNIQUES
A . Introduction
As discussed later on the analysis of pesticide residues. gas-liquid chromatographic
(GLC or GC) techniques in conjunction with selective and specific detectors such as
the electron-caputred and flame photometric detectors are the most widely used instru-
ments and probably the key practical instruments used in routine analysis of low level
pesticide residues in environmental sample . Although G U M S systems can now gen-
erally meet the sensitivity of these selective or specific detectors. they are not as widely
used as the gas chromatography-selective or specific detector system . This is probably
because a sensitive GC/MS system suitable for routine monitoring of trace organics
in the aquatic system has only recently been available . Moreover. the initial cost could
be prohibitive to many laboratories . Therefore. the gas chromatograph with specific
detectors still is a main tool in pesticide analysis . These systems are used complimen-
tary to one another in the few laboratories that are fortunate enough to have both for
trace analysis .
Although "GLC is by far the most widely used method for qualitative and quanti-
tative analysis of pesticide residues,"' this method has certain severe pitfalls. Most
experienced analysts who make use of GLC with the so-called specific detectors, par-
ticularly the most widely used electron-capture detector (ECD), realize that it "does
not provide a positive identification of pesticide in question."' Therefore, after the
preliminary identification based on retention time of an unknown peak against a stand-
ard, further confirmation is needed t o assure its correct identity. It is not uncommon
for another compound or CO-extractivepeak to have the same retention time as that
of the compound in question. Since the analytical data are used for important govern-
ment decisions, for environmental studies, and research, it is of the utmost importance
to verify the validity of the analytical data. The preliminary consideration in a labora-
tory which generates data is to ensure the precise identity of the compounds. In this
regard, many approaches for the confirmation of identity of a compound have been
advocated in the past and many of them are still used in various laboratories. They
are thin-layer chromatography, multi-column GLC techniques, p-values, chemical de-
rivatization in solution and in a solid matrix, infrared spectroscopy, NMR and mass
spectroscopy, and other multi-detection techniques. The following section will very
briefly examine the merits of each approach. (See also Chapter 2 for additional discus-
sion.)
B. Various Approaches o n Confirmation of Pesticide Residue Identity

l. Spectrochromatogram
In order to identify and confirm the residue, Goulden and co-workersz advocated
the so-called "spectrochromatogram" so that the pesticide in question is examined
using several different GLC columns in a gas chromatograph. This approach has not
gained popularity because it requires a major modification to the gas chromatograph,
is confined to one or two pesticides in an extract, and is thus limited in its applicability
to practical routine analysis. Although identification by this approach is quite useful
in some cases, it still cannot be considered positive because as Robinson3 and Reynolds4
pointed out separately, the multi-column GLC technique cannot be regarded as an
independent parameter of identity since it may be shown that various organochlorine
pesticides o n different stationary phases are significantly ~ o r r e l a t e d . ~
2. Thin-Layer Chromatography (TLC)

The inconvenience and lengthy procedure in the paper chromatography technique
contributed to its unpopularity in pesticide residue analysis. The more rapid TLC has
replaced it in an analytical laboratory. TLC is based on similar principles to paper
chromatography with several additional advantages such as a wide choice of absorbent
(coating) materials and better sensitivity. In short, it is more convenient and versatile
than paper chromatography. Thus, TLC has been widely used as a tool in pesticide
residue analysis on a qualitative or quantitative basis with an accessory such as disito-
meter determination. For confirmation purposes, a sample extract after examination
by G C is run side by side with a standard. The thin-layer plate is then developed in an
appropriate solvent or a mixture of solvents. After air drying the developing solvent(s),
the sample side of the plate is covered and the standard side is sprayed with a chrom-
ogenic solution for visualization, with or without UV irradiation. The area on the
sample side corresponding to the standard is scraped off and extracted with an appro-
priate solvent. The extract is reexamined by gas chromatography. If the retention time
of the compound before and after TLC extraction is identical t o that of the standard
then it is interpreted as evidence of its identity. Alternatively, if the concentration of
the compound in question in the sample extract is high enough, the whole plate can
be visualized after chromogenic spraying. For organochlorine pesticides, this is fol-
lowed by UV irradiation. (For more details see Chapter 2). Then, if the R, value (see
definition in Chapter 2) of the compound spot and the standard are identical, it is
inferred that they are likely to be the same compound. This information supplement
and the previous gas chromatographic retention time data are inferred to be verifica-
tion of its identity. However, it is well known that two o r more different pesticides,
metabolites, or CO-extractivescan give similar GLC and thin-layer chromatographic
responses. As examples, dieldrin artifacts are known to be present in corn leaf ex-
t r a c t ~and
, ~ aldrin artifacts are present in cabbage, turnip, asparagus, and certain rub-
ber p r ~ d u c t s These
.~ artifacts are probably due t o the sulfur and sulfur compounds
present in these crops and material. Sulfur has long been known to have the same GLC
responses as aldrin7 on several common GLC columns. Some rubber and petroleum
products also give GLC responses similar to dieldrin and DDT.' These products may
also be present in the surface water and are extracted during analysis. Another exam-
ple9 is the detection of two artifact peaks from certain plastics having identical or
similar analytical responses to o,p'- and p,p'-DDE. They have identical retention times
to the DDE isomers on two GLC columns with different polarity using ECD and mi-
crocoulometric detectors. The p-value from acetonitri1e:petroleum ether partitioning
of one of the artifacts (0.62) and p,p'-DDE (0.58) is also quite close. To complicate
matters, two or more different pesti~ides'~," or m e t a b o l i t e ~ ' ~can
- ' ~ give similar GLC
and/or TLC responses. For example, heptachlor and one of the degradation products
of technical chlordane: 2 - c h l o r o ~ h l o r d a n e ~have
" ~ ~ similar GLC and TLC responses,
whereas a- endosulfan and p,p'-DDE1O.L1 have identical GC retention times using some
commonly used columns. Therefore, even if both G C and TLC data agree with each
other, the evidence is not always sufficient to constitute positive confirmation of the
identity of a compound. Furthermore, both G C and TLC are not considered widely
different techniques since they are based on similar principles. As a result, it is not
uncommon to have an artifact having the same TLC and GC response as an analytical
standard.
3. Multi-Column G L C Techniques
This is one of the more widely used techniques in confirmation of residue identity.
It is simple and quick to use. This may contribute partly to its popularity. By this
technique the compound in question is first analyzed by gas chromatography, using
first a polar phase column, and then reanalyzed using a nonpolar phase column o r
vice versa. If the retention time of this compound is identical to that of the standard
in both cases, then it is inferred to be substantiation of its identity. Occasionally further
verification is obtained by using more than two different columns. A good example
of the usefulness of this approach is the interference of sulfur or sulfur compounds
with aldrin in several less polar mixed liquid phase and single liquid phase columns
but not in more polar 0V-225 column. However, as mentioned previously, this ap-
proach, although quite useful, still cannot be considered positive. A good example is
our experience in the early 1970s when a chlordane artifact was observed in fish and
sediment extracts from Hamilton Bay, Ontario, Canada. This artifact behaves like the
c i s and transchlordanes on various GC columns of different polarity. In this case,
the multi-column technique could not provide confirmation of its identity. This expe-
rience was shared by another laboratory18 on different samples from the same location.
It was through chemical derivatization that the artifact(s) was indicated although the
nature of it is not known. Therefore, the analyst should bear in mind the limitation
of the multi-column approach when it is put in practice.
4. p- Value
Based on the concept of distribution (partition coefficient) of a pesticide residue
between two immiscible solvents, Beroza and B ~ w m a n ' ~proposed
-~' the use of extrac-
tion p-values for the identification or confirmation of pesticide residues. p-Values are
defined as "the fraction of the total solute partitioning into the upper phase." (See
more detailed discussion in Chapter 2, Section IV.) This approach gains some popular-
ity because it is simple, rapid, and found to be practically independent of pesticide
concentration and presence of some CO-extractivesin the ample.'^,^^ Although it was
suggested that one or more pairs of solvents be used for positive identification, "the
additional independent evidence for identity gained by obtaining a second p-value is
very Moreover, p-values are of limited value if more than one compound
with the same GLC retention time is present in the same sample extract. Disregarding
this limitation, the use of p-values for confirmation of residue identity is valuable to
residue analysts.
5. Chemical Derivatization Techniques

One of the most useful procedures for confirming pesticide residue identity is the
chemical derivatization gas liquid chromatographic technique (CD-GLC). By using this
approach, the parent compounds are determined prior to chemical derivatization and
the resulting derivatives examined by GLC to provide specific identification. At pres-
ent, with the exception of some suitable G U M S instruments, this approach is the
other most sensitive confirmative method. A good CD-GLC method will generate de-
rivatives with similar or even higher detector sensitivity as compared to that of the
corresponding parent compounds. Therefore, the CD-GLC technique is extremely use-
ful for those samples which contain insufficient residue for confirmation by other
methods such as TLC or IR, and in some cases even GC/MS. In the case of natural
water samples, as Holden and Marsdenz8 in 1965 pointed out and was quoted by
McCullyz9 "confirmation may only be possible by chemical treatment of a residue,
and further examination by GLC." This is due to the usually low level of the com-
pounds in water. Even today, with sensitive GC/MS instruments and convenient con-
centration techniques such as the use of XAD resin (see Chapters 2 and 3) to concen-
trate large amounts of water samples, this old statement is still generally true for
routine and monitoring laboratories which deal with water samples usually of l- or 2-
liter volumes.
So far, the bulk of research and investigation in the area of confirmation of identity
has been done on organochlorines and only a limited amount of work on other pesti-
cides. Therefore, the greater part of the following discussion will be on 0.c.s.
The following discussion will be separated into two groups, namely, O.C.pesticides
and other pesticides. A general discussion on various derivatization approaches and
techniques for the confirmation of 0.c.s will be presented first, followed by a more
specific discussion on 0.c.s. To facilitate this discussion, the O.C. pesticides are sepa-
rated into groups, although some methods can simultaneously confirm compounds in
more than one group.
For definitions of terminology and general discussions of various analytical tech-
niques mentioned in this chapter, refer to Chapter 2.
11. GENERAL DISCUSSION OF DERIVATIZATION TECHNIQUES FOR

0.C.S
A. Introduction
For confirmation purposes, derivative formation of an O.C. pesticide can be achieved
chemically or photochemically. There are three approaches for chemical derivatiza-
tion, and two approaches for photochemical derivatization. These approaches and
their application so far reported are summarized in Table 1 and Table 2, respectively.
Photochemical reaction for confirmation of pesticide residues has been proposed,30
Table 1
VARIOUS APPROACHES FOR THE
CONFIRMATION OF PESTICIDE
RESIDUES BY DERIVATIZATION
TECHNIQUES
Chemical derivatization
Vapor phase reaction (reaction gas chromatography)
Reaction in solution
Reaction on SM
Reaction column chromatography
Reaction on TLC
Photochemical derivatization
Reaction in solution
Solid state reaction: coating on glass or thin-layer
chromatographic plates for UV irradiation
Table 2
APPLICATION OF VARIOUS APPROACHES FOR CONFIRMATION OF
PESTICIDE RESIDUE IDENTITY
Approaches Reaction type Pesticides
Vapor phase Rx (Reaction GLC) Dehydrochlorination DDT + analog

On-column transesterification Carbamates
Some 0.p.s
Reaction in solution Various types (see Table 3) All 0.c.s
Transesterification Esters of phenoxy and carboxylic
acid herbicides
Reaction on SM DDT + chlordanes
DDT + chlordanes (except hepta-
chlor and its epoxide)
a- and /3-Endosulfans + endrin
a- and /3-Endosulfans + heptachlor
+ endrin
Al2O3/H,SO4/H~O (50 g15 Endrin
m!/ 2.5 ml)
AIzO,/H2SO,/H2O (50 g110 Heptachlor in the presence of PCBs
m1/5 m1)
Photochemical Rx In solution (hexane) Various 0.c.s
On TLC or glass plate Various 0.c.s
but it generally results in derivatives with considerably less ECD responses compared
with the corresponding parent compounds and hence is a much less sensitive method.
Furthermore, two or more degradation peaks from each compound were ~ b s e r v e d . ~ '
Due to the complexity of the degradation solution and the low sensitivity of the
method, this procedure30 has little use in routine analysis. What would the chromato-
gram look like if several pesticides were in the sample extracts? The presence of co-
extractives, even after usual cleanup procedures, would yield additional peaks follow-
ing UV irradiation. However, in 1971, these a u t h ~ r s ~refined
l - ~ ~ this method which is
applicable to routine analysis for foods. The refined method encompasses the separa-
tion of individual eluates from a gas chromatographic column and trapping of the
individual fraction in a TeflonB capillary tube. The trapped component after disso-
lution in solvent was then irradiated and the resulting solution examined by GLC. Each
pesticide examined gave a characteristic pattern of peaks including some unreacted
parent peak. However, this method is limited to high-level pesticide residues in a sam-
ple and is not applicable to ultra-trace analysis as for water samples.
The other approach for generating derivatives is by chemical reactions. It can be
achieved in three different ways (see Table 1). The first is the so-called reaction or
derivatization gas c h r ~ m a t o g r a p h y whereby
~ ~ - ~ ~ high temperature chemical modifica-
tion of compounds occurs at the inlet (packed with solid reagent) of a gas chromato-
graphic ~ y s t e m . ~ ~This
- ~ Ois the most convenient approach most applicable to ultra-
trace organic analysis and with minimum mechanical loss. Unfortunately this approach
has a much narrower scope of application than the confirmation procedures carried
out in solution or on a solid matrix due to the limited amount of chemicals that are
suitable for this application. Hence, the number of reactions possible are limited. For
o.c.s, only the DDT and c i . + c h l ~ r d a n e ~can
~ , ~be
' reacted satisfactorily using
this approach. Furthermore, due to different physical design of various gas chroma-
tographs, not all gas chromatographs can be readily converted into a reaction gas chro-
matograph. An alternative approach42that can be applicable to most makes of gas
chromatographs is to extend the injection inlet outside the gas chromatographic sys-
tem. A commercial or homemade pre-column heated injector controlled by its own
thermocouple is screwed onto the inlet system of the GLC. It is furnished with a glass
tube for the appropriate catalyst. A septum is screwed on the top of the micro reactor
for injection and the carrier gas passes through the reactor before entering into the
column. In either of the above cases, if the design of the modification has no provision
to prevent the carrier gas from going through the pre-column or inlet reactor to the
regular GLC column and to the detector when the systems are not in use, the GLC
performance can be adversely affected, due to leaching of materials from the pre-col-
umn under high temperature and constant sweeping by the carrier gas.
Although chemical reactions carried out in solution have been applied to colorimet-
ric development since the early stages of pesticide residue analysis in the 1950s, its
application to residue analysis is a more recent development. The first papers on the
subject of CD-GLC, to the knowledge of this author, appear to be that of Hammence
and in Britain and of Sans7" in Canada. However, it was not until 1969 that a
Canadian group began to investigate systematically the CD-GLC techniques for the
positive identification of pesticide residues and some degradation products encoun-
tered in routine analysis. Many organic reactions have been utilized for this purpose.
They are summarized in Table 3.
B. Solid Matrix (SM) Derivation Techniques

Recently, in order to further simplify and increase the detection sensitivity of the
chemical derivation procedures, a new chemical derivation approach for confirmation
of pesticide residues has been developed. This approach, termed solid matrix (SM)
derivation techniques, involves derivation in a solid matrix instead of the previously
reported derivation procedures carried out in solution.
Briefly, sample extracts usually after concentration are adsorbed on a small column
such as disposable pipets filled partially with a solid matrix such as alumina impreg-
nated with appropriate chemicals. Depending on the reactions, the columns are left at
room temperature or at elevated temperatures for varying periods of time. Then, each
column is eluted with an organic solvent to obtain the derivative formed.
Although the SM derivation technique for the confirmation of pesticide residue iden-
tity is only a recent endeavor, reactions on a solid matrix were r e p ~ r t e d some
~ ~ - time
~~
ago in synthetic chemistry mostly on more active molecules. For example, the work
of DevS5involves the opening of methyldialsyl-1,2-epoxides,particularly of the hum-
mulene epoxite analogs, by shaking it with acidic alumina in organic solvents.
Another example of SM reaction is the dehydrofluorinationS6 at C-5 in some satu-
rated 3-keto steroids t o give the A4,=-3keto steroids, accomplished by passage of the
saturated steroid through neutral alumina.
Table 3
TYPE O F REACTIONS USED FOR T H E CONFIRMATION O F
PESTICIDE RESIDUES BY CHEMICAL DERIVATIZATION - GAS
CHROMATOGRAPHIC TECHNIQUES (CD-GLC)
Type of reaction Reagent Pesticides
Dehydrochlorination KOH or NaOH DDT and analogs

t-BuOK DDT, analogs, and chlordanes
Reductive dechlorination CrC12 Heptachlor + endrin, endrin
metabolites, 0.p.s
Addition Bromination Aldrin
Chlorination Aldrin
t-BuOCl/t-BuOH Aldrin + heptachlor
t-BuOCl/Ac O H Aldrin + heptachlor
Peracids Aldrin + chlordene
Epoxide rearrangement H2304 Endrin + dieldrin
H,S04/Ac20 Endrin + dieldrin
BCl,/,-2CIEtOH Endrin + dieldrin
HCl/ZnCl, Endrin + dieldrin
Oxidation CrO, DDE + endrin + heptachlor
Intramolecular Rx (base-catalyzed) KOH/ROH a- + p-Endosulfans
t-BuOK Endrin cyclic ketones (degra-
dation products of endrin)
Intramolecular Rx (acid-catalyzed) H,SO,/AI,O, (on Endosulfans
SM reaction)
H,SO, in solution Endrin
H,S04/A120a Endrin
(SM reaction)
Cleavage of sulfite moiety LiAIH, followed Endosulfans
by silylation or
acetylation
Ac,O/H,SO, (in Endosulfans
solution)
Ac2O/H2SO4/ Endosulfans
A120, (SM reac-
tion)
In the elimination of the tosyl group (TsO-) of labile compounds containing tosylhy-
drazine derivative, M ~ c h o w s k iobtained
~~ 80 to 100% yield of the diazoketones by
stirring the compounds in fairly strong basic alumina (pH 10 to 10.5) suspended in
methylene chloride or ethyl acetate. This replaces the often low yield of a heteroge-
neous system of dilute aqueous NaOH and an organic solvent for the same purpose.
Meinwald et al.58obtained an interesting diazoketone by such SM reaction.
In another e ~ a m p l e , ~
alumina
' impregnated with potassium hydroxide was used for
the in situ generation of dichlorocarbene or dibromocarbene which is a reactive inter-
mediate to attack double bonds, for example:
Due to the low yield of both reactions the generation of the halocarbene from halo-
form and the subsequent formation of the gem-dihalide, this reaction was unsuccess-
fullys9applied to the confirmation of aldrin on a SM, or by reaction in a slurry.
In the application and development of SM derivation techniques for the confirma-
tion of pesticide residues identity rather than the often-used procedures of stirring the
reactant with alumina in synthetic chemistry, the reactions were carried out at various
temperatures in a column packed with alumina and impregnated with the appropriate
chemicals. This approach has been used for macro scale (preparative) as well as for
residue level reactions of pesticides t o form derivatives. Silicic acid or FlorisilO was
not used in the SM derivatization technique because alumina was found to be superior,
possibly due to some surface catalytic effect of the latter.
The general procedure for the SM derivation and confirmation of pesticide residue
identity is as follows:
A suitable aliquot of sample extract such as 1 to 2 m1 of fish or sediment extract (more for water sample
extracts) is evaporated to approximately 200 p1 with toluene or mineral oil as a keeper. The concentrated
solution is then applied to a reaction column packed with l " or 2" of SM. About 100 p1 of organic solvent
such as hexane or benzene is added to distribute the extract on the upper portion of the column. After
reaction, usually 1 hr at room temperature or elevated temperature, the column is eluted with a solvent and
the eluate, with or without concentration, is then examined by GLC.
C . Types of Solid Matrix Developed for Pesticide Confirmation

There are several types of SM so far developed for confirmation purposes (Table
4). They are briefly described below:
1. Alumina/KOH/H,O (200 g neutral or basic/20 g/15 mP).60 It is suitable for the

confirmation of DDT type compounds (without heat) and chlordanes (with heat).
2. Alumina/potassium tert/butoxide (Al,O,/t-BuOK: 100 g/20 g).60It can also be
used in the same way as SM No. 1 but is not suitable for heptachlor and hepta-
chlor epoxide.
3. Alumina/acetic anhyldride/sulfuric acid (A120,/Ac20/H2S0,: 50 g/10 m115
m1).61 It is only suitable for the confirmation of endosulfan (a-and p-isomers)
which are converted to the same diacetate. The keeping quality of this SM and
the sensitivity of the method using it are relatively inferior to the one discussed
below (SM No. 4).
4. Alumina/sulfuric acid (A1203/H2S04:50 g/10 mL).62,63 It was found to be an
exceptionally excellent SM for routine confirmation of endosulfans in terms of
(1) its good stability over several months, (2) excellent reproducibility and sensi-
tivity obtained, and (3) its cleanup power to destroy or eliminate sample co-ex-
tractives which may cause analytical interferences. In addition to its application
for endosulfans, it also can be used for confirmation of heptachlor residue.
5. Aluminium/sulfuric acid/water (Al2O3/H,SO,/H2O : 60 g/5 m1/21/2 m1).64 It
was designed for the convenient confirmation of endrin residues in routine appli-
cation. This reaction can be easily carried out on a few dozen samples with min-
imum attention even by inexperienced personnel. The small amount of water is
Table 4
SUMMARY OF SM TYPES AND APPLICATION TO IDENTITY CONFIRMATION OF
PESTICIDE RESIDUES
Reagent Reaction condition Application Product Ref.
Al,O,/KOH/H,O Room temperature over- All DDT type + cischlordane Dechlorinated products 60
200 g/20 g/ 15 m1 night
7S°C, 1-3 hr As above + cis and trans Dechlorinated products
chlordanes, heptachlor and its
epoxide
Al,O,/t-BuOK/ t-BuOH 7S°C, 1 hr All DDT types + cis and Dechlorinated products 60
100 g/20 g/75 m1 transchlordane only
A1203/A~~O/H~S04 100" 2 S0C, 2 hr a- and p-endosulfans Endosulfan diacetate 61
50 g/10 g/5 m1
A1203/H2S04 100" + S a c , 1 % hr a- and p-endosulfans Endosulfan ether 62,63
50 g/10 g Heptachlor l-Phenylchlordene
AI,0,/H2S0,/H20 Room temperature, 1 hr Endrin Endrin ketone 64
50 g/5 m1 /2-% m l o r overnight
A1203/H2S0,/HI0 95-10O0C, 1% hr Heptachlot I-Hydroxychlordene 65
50 g/10 m1/5 m! Endrin Endrin ketone
Volume I 93
Table 5
SUMMARY OF KEY ANALYTICAL STEPS TO PROCESS A SAMPLE FOR
0.C.s AND PCBs RESIDUE ANALYSIS
1. Comminution of solid samples (if necessary)
2. Extraction with organic solvent
3. Clean-up
a. Liquid-liquid partitioning
b. Column chromatography (e.g., FlorisilO column)
4. Determination by ECD-GLC (electron-capture detector-gas-liquid chromatography)
5. Confirmation of identity
6. Establishment of quantitative validity (in-house and inter-lab quality control activities)
Adapted and modified from Reference 329.
necessary to deactivate the SM so that minimum amounts of nonpolar solvent

could be used for complete elution of the more polar derivative (i.e., endrin ke-
tone). On the other hand, too much water will decrease the yield of the derivative.
6. Alumina/sulfuric acid/water (50 g/10 mP/5 rnP)."' This was used for the confir-
mation of heptachlor by converting it to l-hydroxychlordene. Endrin can also
be confirmed since it is also converted to the endrin ketone; however, the yield
is about 5% lower than that obtained from using SM No. 5 above. The present
SM method is most suitable for the confirmation of a small amount of heptachlor
in the presence of a large amount of PCBs in the sample.
The use of microcolumn-SM techniques for transesterification of five methyl esters

of herbicidal acids to the corresponding 2-chloroethylesters was found66to be feasible
with an overall 60% yield. However, refinement of the experimental conditions to
optimize this transesterificatio has yet to be carried out.
Another version of the SM approach is to react sample extract on an alumina thin-
layer chromatographic plate."' This procedure involved the reaction of heptachlor res-
idue on thin-layers of neutral alumina to produce l-hydroxychlordene. It appears that
not all neutral alumina give the same results since the authors observed that the "best
results are obtained on strips of ready made plates such as alufoil Al,O,E, Merck
catalog number 5550/0025." It could be some unknown minor constituents present in
alumina of various makes that increase or decrease the yield of the reaction. This
method can be used to confirm heptachlor in the presence of large amounts of PCBs
or polychlorinated naphthalene using a more polar developing solvent.
111. ORGANOCHLORINE PESTICIDES ( 0 . C . S )
Before the specific discussion on CD-GLC techniques, it should be realized that for
the analysis of O.C. residues a sample is extracted and the sample extract cleaned up
by liquid-liquid partitioning or column chromatography or both to remove interfering
CO-extractivesfrom the analytes (Table 5). Detailed discussion on sample treatment is
presented in Chapter 2 for pesticides in general and in Volume 11, Chapter 1 for 0.c.s
specifically.
Quite often in multi-residue analysis, the column chromatographic cleanup proce-
dure also groups the 0.c.s into several fractions to facilitate subsequent GC/ECD iden-
tification and determination. After this preliminary separation, the identity of each
pesticide in individual fraction can be confirmed by a CD-GC technique or other tech-
niques discussed previously. The key steps to process a sample for GC analysis are
summarized in Table 5. FlorisilO column cleanup procedure is widely used for multi-
residue 0.c.s analysis. It is recommended or included in standard analytical methods
Table 6
FLORISIL COLUMN FRACTIONATION OF PESTICIDES
Sample extract
I
~ l o r i s i l @column
6% Diethyl ether in petroleum

+ ether fraction
Petroleum ether fraction
- -
or-BHC, p,pl-DDE, aldrin o,pl-DDT, lindane,

mirex, photomirex, PCBs cis- and trans-chlordanes,
p,p'-methoxychlor, heptachlor
epoxide
-
Mini-charcoal/foam column
15% Diethyl ether in

petroleum ether fraction
Endrin, or-endosulfan,
dieldrin
Cyclohexane Benzene
fraction fraction
50% Diethyl ether in
petroleum ether fraction
photomirex, or 100%CHC1,
heptachlor,
aldrin
manuals such as the FDA PAM,68 WQB Analytical Methods Manual," and AOAC
Methods of Analysis.'O Therefore, in the following discussion on CD-GLC technique
for a particular o.c., the primary concern is the effect of the derivatized procedure on
other 0.c.s that can be present in the eluant (fraction) after Florisila column cleanup
of the sample extract. The various fractions from the FlorisilF column cleanup pro-
cedure are summarized in Table 6. It should be referred to whenever a derivatization
procedure is discussed. However, when data are available, the effect of a derivatization
procedure on those 0.c.s eluted in different fractions than the O.C.in question will also
be briefly discussed to assist those who may use a different fractionation and cleanup
technique.
A. DDT Group
Among various chemical systems used in reactions for confirmation of identity as
well as for the analysis of organochlorine pesticide residues in agricultural commodities
and in environmental samples, reaction of DDT and similar compounds with alakli
are the most widely used and investigated. Even before gas chromatography was avail-
able, several in the 1950s used alkali to convert DDT-type pesticides such
as methoxychlor and perthane to the respective olefins which were further treated with
certain chemicals to form complexes for micro-calorimetric determination.
Volume I 95
Table 7
STRUCTURES O F DDT AND ANALOGS AND THE RESPECTIVE OLEFINS
FROM DEHYDROCHLORINATION
cl-C-cl Cl-c-cl
I
Cl p,p'- DDE
p,p'- DDT
i
H
~ o ~ / e t h ~ l eglycol
ne
DDM DBP
Cl DDMU
p, p'- DDD
H
DDNU
DDMS
In the early 1960s Kein and Watts7s wrote of the need for additional confirmatory
evidence to determine small amounts of DDT in butter by GLC/ECD. These auth-
orS75-76
proposed the determination of the olefins of DDT and related compounds as
a confirmatory evidence. Thus, after GLC determination of the parent compounds
such as p,p'-DDT, perthane, and p,p'-DDD, the parent compounds were converted to
the corresponding olefins (Table 7) by refluxing with NaOH in ethyl alcohol. The ole-
fins were re-examined by GLC. Sample extracts and the appropriate standards were
treated the same way for comparison.
With the advent of the electron-capture detector (ECD) in gas chromatography, de-
hydrochlorination of DDT and related compounds to generate the more ECD sensitive
olefins became popular. There are many variations reported in the literature on the
dehydrochlorination procedures for analysis or for confirmation purposes. Some ap-
plications of the alkaline dehydrochlorination reaction for the conversion of DDT and
analogs to the respective olefins as confirmatory tests are summarized in Table 8. In-
variably an alcoholic solution of potassium or sodium hydroxide, and to a much less
extent sodium methoxide in alcohol, under different experimental conditions were
used. All these procedures gave good results because of the ease with which these com-
pounds are dechlorinated in excellent yield, particularly in the case of DDT to the
respective olefins. Also, the olefinic products, notably p,p'DDE are very stable under
the conditions used. Therefore, there is little fear of further reactions in the alkaline
system once they are formed even though the time and alkalinity for these dechlorina-
Table 8
APPLICATIONS O F ALKALI DEHYDROCHLORINATION FOR DDT AND
ANALOGS
Reaction Known
Compounds Reagents conditions interference Ref.
p,p'- DDT 2% NaOH in ethanol ( l 0 m!) Reflux, 20 min Chlordanes 75

P,P'- DDT, 2% NaOH in ethanol ( l 0 m!) Reflux, 20 min Chlordanes 76
o,p'- DDT, p,p'- DDD,
perthane
p,p'- DDT and DDD, 0.5NAlcoholic KOH, (0.5 m!) Room temp., 5 min Chlordanes 77
o,p'- DDT
p,p'- DDD and DDT, K X O , solid Flash heater (240°C). Chlordanes 39
methoxychlor Rx C L C
p,p'- DDT + DDD, 2 % KOH in 95% ethanol, 5 m l 77"C, 15 min Chlordanes 78
o,p'- DDT
p,p'- and o,p'- DDT 5 % NaOH in ? m! Reflux, 30 min Chlordanes 79
p,p'- DDT, o,p'- DDT, Anhydrous NaOMe Shake with l m1 sam- Chlordanes 80
p,p'- DDD, methoxychlor ple extract in hexane
(1 min or 20 min)
room temp.
p,p'- DDT, o,p'- DDT, NaOH + KOH o n gas Chrom Alkaline pre-column Chlordanes 81
p,p'- DDD, methoxychlor, Q (200"C, ?) Rx GLC
perthane
p,p'- DDT; o,p'- DDT, 2 % KOH in 95% ethanol, ? m1 80°C, 12 min Chlordanes 82
p,p'- DDD, methoxychlor
p,p'- DDT and DDD chlor- t-BuOK/ t-BuOH Reflux (82-85°C) 30 - 13,14
danes 200 mg/2 m! min
o,p'- and p,p' - DDT NaOH, p H 8 30°C, 10 min Chlordanes 83
o,p'- DDT; o,p'- DDD per- t-BuOK/ i-BuOH 50°C or reflux 30 min - 84
thane, rnethoxychlor 200 mg/2 m!
p,p'- and o,p'- DDT, DDD, 2% KOH in 95% ethanol, 2 m! Reflux at 100°C 15 Chlordanes 85
perthane, methoxychlor min
(and other pesticides)
p,p'- and o,p'- DDT, DDD 2% KOH in ethanol ( l m!) Reflux and heat ( l 5 Chlordanes 86
perthane, methoxychlor min) to 0.2 m l
DDT, DDD isomers, per- AI,O,/KOH Room temp. or 75'C, 60
thane, methoxychlor, 200 g/20 g 1 hr and 3 hr
chlordanes
A120,/ t-BuOK 75"C, 1 hr Chlordanes 60
100 g/20 g
tion steps are somewhat excessive. In the case of DDE, for example, extremely drastic
conditions are required to degrade it - such as heating with excessive alcoholic potas-
sium hydroxide solution in a sealed tube at 150 to 160°C for 20 hr to yield the di-(p
chloropheny1)-acetic acid.lO' Because of the various experimental conditions advocated
by numerous authors for the dechlorination reaction mentioned above, Young and
Burkes6 reinvestigated this reaction using a refluxing alcoholic potassium hydroxide
"to arrive at optimum and convenient parameters of alkali treatment ... for simple
micro scale application in multi-residue analysis" applicable to p,p'-DDT, o,p'-DDT,
p,p'-DDD, o,p'-DDD, methoxychlor, and perthane. This work is complimentary to
that of KrauseE5who did a thorough study on the quantitative dehydrochlorination of
perthane by alcoholic potassium hydroxide including its effect on many other pesti-
cides.
In contrast to all those procedures mentioned above, Chau and Cochrane in 196913,'"
used a stronger alkaline system, i.e., a refluxing solution of t-BuOK in t-BuOH for
the following reasons. First, unlike all the dehydrochlorination procedures reported,
this stronger system not only dehydrochlorinates DDT and analogs almost quantita-
tively to the respective olefins but also converts c i s and transchlordanes, nonachlor,
heptachlor, and its epoxides to their corresponding derivatives in good yield suitable
for subsequent G C analysis. In other words, the scope of the dehydrochlorination
approach is considerably enlarged. Second, this procedure can destroy more effectively
any coextractives that may still be present in the sample extracts. For "dirty" samples,
such as some sediment samples, and wildlife and fish samples, this characteristic was
found to be beneficial to interpretation of GC chromatograms. But most important
of all, this drastic system is superior t o the milder system using KOH, NaOH, and
even NaOMe for the confirmation of DDT type compounds if the more stable chlor-
dane group is also present. This is due to certain peak overlap of the expected olefinic
products from DDT and its analogs with peaks from unreacted or partially reacted
chlordane type pesticide already present in the sample extracts. For example, peaks
such as o , p D D E and DDMU which are alkali dehydrochlorinated product of o,p'-
DDT and p , p D D D , respectively, are not sufficiently resolved from the peak of hep-
tachlor epoxide and transchlordane in many commonly used GC columns. These
chlordane pesticides are only partially reacted even at refluxing temperature in alco-
holic potassium or sodium hydroxide. The use of the stronger alkaline system of t-
BuOK/t-BuOH therefore eliminates this chromatographic interference because all the
respective products have different retention times in several GC columns to enable
their distinction.
In addition to the common alkali, Milesa7used DBU (1,5-diazobicycle (5.4.0.) un-
dec-5-ene) t o convert o,p'-and p,p'-DDT to the respective olefins. However, using this
more exotic compound offers no significant advantages.
T o simplify the dehydrochlorination procedures, a SM technique for confirming the
DDT group and chlordane group was later d e v i ~ e d . ~As" discussed earlier, the reaction
was carried out in a micro-column, in this case packed with alumina impregnated with
either KOH or t-BuOK. After reaction, only 2 m1 of benzene is required for complete
clution of the derivatives for GC analysis. The SM derivatization technique is particu-
larly suitable for samples such as water containing extremely low levels of pesticides
because the mechanical steps required when reactions are carried out in solutions are
minimized by the SM procedure. Also, the volume of eluate in this case is only 2 m1
which facilitates further concentration if required.
A further remark on the dehydrochlorination of DDT to DDE as a means of confir-
mation is that generally, p,p'-DDT occurs together with its metabolite DDE, which
can interfere with the confirmation of DDT by dehydrochlorination unless the DDE
has been preseparated from the DDT. Generally, this preseparation is the common
practice in multi-residue analysis. In many laboratories, column chromatography, for
example, Reynolds' FlorisilB procedures8 is used for sample cleanup and fractionation
of to facilitate GC analysis and interpretation (see Table 6 ) . There-
fore, small amounts of DDT in the presence of large amounts of DDE can still be
confirmed by the dehydrochlorination procedure since these two compounds are dis-
tinctly eluted in different fractions.
At the time of preparing this manuscript, dehydrochlorination reaction is the only
practical approach for the confirmation of DDT and analogs to meet most of the
criteria for good confirmation procedure (see Section V). Other reactions such as de-
chlorination by chromous chloride (CrC1,),90 nickel boride (NiB),9'.92or sodium na-
phthalenide (Na Nath)93are not applicable for this purpose because a number of deriv-
atives are generated and the derivatives have considerably less or little ECD response.
Also, the number and yield of certain products by these reactions varies greatly with
time of reaction and is not easy to control. For example, in the case of CrC12 reaction
at 60°C for the first 30 min or so, p,p'-DDT gave two major products: trans-DCS
(transdichlorostilbene) and p,p'-DDD. The DDD was further reacted by CrC12 to

DDNU (1,l '-bis[pchlorophenyl] ethylene), DDMU (1,l'-bis[pchlorophenyl]-2-chlo-
roethylene), and transDCS (trans-p,p'dichlorostilbene). This unstable transisomer is
further isomerized to the cisisomer upon standing in s o l ~ t i o n The . ~ ~
other
~ ~ two
~ ~ ~ ~
reductants, NIB and Na Nath, are more drastic and dechlorinate more chlorine atoms
to give products with much lower ECD responses.
Other approaches for confirmation of DDT and analogs as well as other O.C. pesti-
cides involve UV photolysis in s ~ l u t i o n ~ O or- on
~ ~ thin film (solid ~ t a t e ) . ~Thermal
~-~'
decomposition (pyrolysis at 900°C)99was also investigated. All these approaches re-
quired preseparation of pesticides into single components before UV radiation or pyr-
olysis; otherwise, in most cases the GC chromatogram of the reacted fraction would
be too complicated to be interpreted to have any practical use. This preseparation step
is the most important step for successful application of either approach. Only the pro-
cedure by Hoffman et for liquid phase photolysis incorporates a detailed and
workable preseparation procedure which involves trapping of individual eluant from
GC column prior to UV irradiation in hexane. TLC or column chromatography as
suggested96 does not have sufficient resolution for clear-cut separation to isolate the
compound free from other 0.c.s and sample CO-extractivesthat may be present in the
same sample extract. These materials can also form UV degradation products; conse-
quently, the chromatogram would be messy and result in more confusion rather than
provision of substantiation to the identity of the pesticide in question.
Between the l i q ~ i d - p h a s e ~ O
and
-~~
the solid-phase p h o t o l y ~ i s in
, ~ ~general, "Liquid-
phase photolysis favors formation of fewer products in higher yield."96 Although it is
true that the solid-phase photolysis technique "provides more definitive degradation
patterns"96 for single and pure pesticide, it is undesirable in practice since it is impract-
ical and difficult to isolate a single pesticide from a mixture and free of unknown
sample CO-extractives. Furthermore, the peak pattern and yield of products by solid-
phase photolysis could vary with film thickness and could be more irreproducible de-
pending on the dexterity of the analyst to generate the film for UV exposure. As
pointed out by K a ~ f m a n n "Both , ~ ~ W i ~ h m a n nand~ ~Lindquist9' had reported for-
mation of a coating on the crystals of DDT. Fleck'00 later reported that a deposit
appeared to form on the outside of crystals of DDT when they were irradiated and
that the deposit appeared to exert a protective action on the intact pesticide." Thus,
if DDT were dissolved in an organic solvent, it was degraded more rapidly.97 This
phenomenon may explain why Wichmann et al.,98who used large crystals for irradia-
tion, observed no effect on DDT and DDE whereas in the case of Lindquist et al.,97
the crystals were so small that they were entirely degraded before a protective coating
was formed. Therefore, a uniform film is necessary for reproducible results and effec-
tive degradation in the case of solid-phase photolysis.
In view of the above discussion, for the confirmation of DDT and other pesticides,
the technique of Kaufmann et a1.30-33is chosen over that by solid-phase photolysis96
in terms of sensitivity and practicality. However, even Kaufmann's procedure33 is not
sensitive enough for most environmental application. This is because in both cases,
considerable amounts of the parent 0.c.s investigated were not degraded, particularly
in the case of the DDD isomers, where most (80 to 90%) of the parent compounds
remain unchanged after 30 min96 or 2 hr33 irradiation. The low yield of derivatives
plus lower relative ECD response as compared to parent compounds make the UV
photolytic method not a sensitive confirmatory test.
Compared with chemical reactions in solution or on solid matrix these UV tech-
niques are also less convenient to carry out. Thermal decomposition of pesticides, par-
ticularly p y r o l y ~ i st o~ ~
gaseous components such as CO, CO,, Cl,, HCl, or 0 2 , can at
best be considered only as a screening method. It is not a practical confirmatory pro-
cedure for most applications due to lack of specificity and sensitivity.
Volume 1 99
DDT and its analogs lend themselves nicely to reaction GLC. The general advantages
and disadvantages of this technique have been discussed previously. In particular, by
the method of Miller and well^,^' except cis-chlordane, heptachlor, and its epoxide,
other chlordane type pesticides are partially or not reacted under the condition speci-
fied. The unreacted and partially reacted chlordane type pesticides can interfere with
the olefinic products of DDT and its analog because these have similar retention times
in many commonly used GLC columns This is similar to the previously discussed situ-
ation when a milder alkali, i.e., KOH, NaOH, or even NaOMe, rather than t-BuOK,
is used for dechlorination of DDT type compounds when chlordane pesticides are also
present. With the exception of heptachlor and DDE, these two groups of pesticides
are usually eluted in the same fraction from most cleanup column68-70,88 (see also Table
6) and are difficult to separate them. Therefore, if DDT and analogs are present with
significant levels of chlordanes in the same sample extracts, this reaction GLC tech-
nique may not be applicable for the confirmation of DDT type compounds.
The conversion of p,p'-DDE by CrO, to p,p'-dichlorobenzophenone as reported re-
spectively by Crist01"~~"~ and Stephenson et a1.'04 in 1945 to 1946, had been applied
t o the analysis of p,p'-DDE as early as 1957 by Gunther and Blinn.loSSans78is proba-
bly the first person to apply this reaction for the confirmation of DDE at residue scale
using ECD/GLC. Unfortunately, ECD has a considerably lower response towards this
benzophenone compared with the parent compound and hence limits its applicability
t o certain environmental samples such as natural waters. Also, DDD if present must
be separated from DDE by column chromatography (Table 6) before the reaction is
carried out because DDD also can be oxidized to the same phenone'04 as DDE.
B. Endrin
It is well known that endrin can be isomerized thermally,'06 photochemically,107-'09
and c h e m i ~ a l l y " ~ -by
" ~ acids (Lewis and mineral acids) (see Table 9). At present, there
are only two types of reactions used for its confirmation. One is based on the acid-
catalyzed intramolecular isomerization as originally reported by Bird et al. in 1961."'
Because endrin reacts so readily under acidic conditions, virtually any acids can effect
this isomerization. Six procedures based on this reaction have been reported (Table
10) for confirmation of endrin residue. The second type of reaction was the consecutive
isomerization and reductive dechlorination by acidic chromous chloride solution (Ta-
ble 10) based on the original finding of Chau et al.'".'20-'24These procedures are sum-
marized in Table 11.
1. Acid-Catalyzed Zsomerization Procedures

The six procedures, which are all based on acid isomerization so far reported in the
literature, have their advantages and disadvantages. Depending on the circumstance
and sample type, one procedure could be more desirable than the other. For example,
in Table 10, the first listed method (sulfuric acid in solution) is the most efficient
among those reactions carried out in solution (Table 10, l a to lb) in destroying co-
extractives and has very low reagent blank. The procedure is also more convenient and
simpler to use. Furthermore, complete conversion of endrin to the ketone can be
achieved in a short time (5 to 10 min) at room temperature. In contrast, the other
procedures using either Ac20, HCl, or boron trichloride (Reactions l b , lc, and Id,
Table 10) require heating. Heating is not necessary to convert endrin to endrin ketone,
but the procedure is so designed t o react with dieldrin also which requires heating.
Also, in comparison with the sulfuric acid procedure, these procedures are less suitable
for water samples due t o higher blank values, unless the reagents are vigorously puri-
fied. In spite of the desirable advantages of the sulfuric acid method, there is a major
disadvantage. That is, if dieldrin is also present in the sample, it also reacts to give
Table 9
MOLECULAR ISOMERIZATION OF
ENDRIN
Endrin Hexachloro pentacyclic

ketone
Reagents Ref.
UV irradiation of solid
Thermal reaction
Lewis acids
Other acids
HOAc/H2S04
Cr 0,
Sulfanilic acid
H S O , (in sol.)
HCI
HCl/ZnCI,
Table 10
METHODS FOR CONFIRMATION OF ENDRIN
Ref.
(1) Acid-catalyzed intramolecular isomerization
(a) H2S0, (in soln) 14
(b) AC,O/H,SO, (in soln) 14
(C)HCf /ZnCI, (in soln) 112
(d) BC1,/Cl (CHI), OH (in soln) 119
(e) H2S0,/A1203 (on sm) 16,154
(f) H2S0,/AI,0, (on sm) 65
(2) Chromous chloride (CrCl2)reaction

(a) Chemistry and mechanism 15,120,121,122,123
(b) Confirmation procedure 17
Endrin
several peaks, one of which has a retention time just slightly before that of the endrin
ketone in many commonly used GLC columns such as SE-30/QF-1 column. Although
the ECD response of this and other dieldrin peaks are considerably lower than that of
endrin, the presence of dieldrin if in sufficient quantity will interfere with the confir-
mation of endrin. This overlap problem has been overcome by using either 15% QF-l
Table 11
SUMMARY OF CHEMICAL CONFIRMATION PROCEDURES FOR ENDRIN
AND DIELDRIN
Reaction Known
Endrin, dieldrin 0.5 m! (1:2, HBr Ac,O) room temp., 30 min

Endrin 4 m1 (15 g CrO, in 50 m! 77°C. 12 min
HOAc)
Endrin, dieldrin 0.5 m1 (0. l g ZnCI, 2 m! IOO°C, 10 min
H C l , 25 m1 abs. EtOH)
Endrin 0.5 m1 H,SOn room temp., 10- 15 min Dieldrin in some 14
GC column
Endrin, dieldrin H2S04/HOAc 100"C, 30 min -
Endrin (heptachlor) Acidic CrC12 55-60°C. 45 rnin I5
sol. in acetone 17
Endrin A120,/H2S0,/H,0 room temp., 1% hr - 64, 118
(l00 g/10 m1/5 ml)
Endrin (heptachlor) A1203/HzS04/HzO
(50 g/10 m1/5 m!)
Two endrin photode- t-BuOK/ t-BuOH followed
gradation products by silylation or acetylation
Endrin, dieldrin BC1,/2-Cl EtOH 90°C, 2 hr
90°C, 10 rnin (endrin
only)
Endrin, dieldrin HCl/Ac,O (20 m l / 10 ml) room temp., 30 min.
(endosulfans, hepta-
chlor epoxide)
and 10% DC-200 or better still a "11%" OV-17/QF-1 Gas Chrom Q column for on-
column resolution. The "1 1%" OV-17/QF-1 column from Applied Science Labora-
tories, Inc. is, in fact, a 1.8% OV-17/4% QF-I column which we also used successfully
in separating 14 0.p.s isothermally (see Volume 11, Chapter 2 on 0.p.s.). The explana-
tion of this "1 1%" OV-17/QF-1 is discussed in Volume I, Chapter 2.
Alternately, the H2S04/Ac20procedure (Table 10, lb) can be used to confirm en-
drin and dieldrin simultaneously when both of these pesticides occur together in the
sample extract. Endrin will be derivatized to the same ketone (Table 9) and dieldrin to
a trans diacetate as the main product. These derivatives are easily resolved on many
GC columns. However, this procedure is not as desirable for some sample extracts as
the sulfuric acid method in terms of reagent blank level and the generation of extra-
neous peaks from some sample CO-extractives.
The American methods (Wiencke and B ~ r k e , "Woodham~ et al.11z),similar to the
H 2 S 0 4 / A C 2 0method, also produce derivatives of endrin and dieldrin with distinctive
retention times in many GLC columns. The method by Woodham et al. is one of the
most sensitive tests for dieldrin published so far, although it is still not sensitive enough
for most of the environmental samples, except possibly certain wildlife samples. Si-
multaneous confirmation of dieldrin and endrin can also be achieved by the acetic
anhydride-sulfuric acid method,'" but this method produces a product of low GC sen-
sitivity for dieldrin, which greatly limits its usefulness for the confirmation of dieldrin
residue. Also, it has a considerably higher reagent background than the sulfuric acid
procedure.
In view of the general advantages of SM derivatization techniques (see earlier dis-
cussion), the use of this technique was extended to the confirmation of endrin residue
based on the idea of sulfuric acid isomerization in solution (Table 10, l e and If). This
approach offers three major advantages. First, due to the adsorbing power of this SM
and its effectiveness to eliminate and minimize sample CO-extractives,this SM proce-

dure is especially ideal for water samples which often require concentration to a small
volume (0.5 to 1 m!) before GC examination. This feature was also found advanta-
geous for "dirty" environmental extracts such as some fish and sediment extracts.
Second, due to the design of the procedure and the intrinsic character of the tech-
nique, confirmation of endrin residues in several dozen samples can be carried out
simultaneously within a short period of time even by an inexperienced technician. The
minimum mechanical steps involved and the stability of the SM make the procedure
almost foolproof and minimize the chance of variations due to mechanical manipula-
tion in extractions, transferring, etc. as in the procedures which are carried out in
solution.
Third, this procedure greatly minimizes the interference from dieldrin and could
tolerate the presence of a larger amount of dieldrin without its interference for the
confirmation of endrin.
2. CrCL Reaction
A second type of reaction used for the confirmation of endrin resulted from original
findings of Chau (Table 10).12.15-17.23.118.'20
It was based on the consecutive acid-cata-
lyzed isomerization and reductive dechlorination in acidic chromous chloride solution.
It should be emphasized that in order to convert endrin almost exclusively to the pen-
tachloro product, the chromous chloride solution used must be on the acidic side at a
p H below 4.lZ0If the chromous chloride solution is not acidic enough, as is the case
when dissolving purified solid chromous chloride in water, endrin is converted to mul-
tiple products including the pentachloro ketone mentioned above. If the chromous
chloride solution is not acidic enough a drop of dilute hydrochloric acid (1:3) added
to the chromous chloride solution used for each reaction is sufficient to provide
"clean" conversion of the endrin residue to the pentachloro ketone.
The sensitivity of this method is comparable t o that from acid-catalyzed isomeriza-
tion by sulfuric acid, boron trifluoride, or hydrochloric acid mentioned above. Due
to the low reagent blank of chromous chloride, concentration of reaction extracts to a
small volume before GLC injection is possible. This procedure can confirm endrin and
heptachlor simultaneously if they have not been pre-separated by column chromatog-
raphy such as the FlorisilB column (see Table 6 ) . The most advantageous aspect of
this procedure is that there is no interference from dieldrin as encountered in the sul-
furic acid catalyzed method, although dieldrin reacts appreciably under the experimen-
tal conditions.
In conclusion then, if dieldrin is not present in the sample or a suitable G C column
is used (see above discussion), the confirmation of endrin is best carried out by using
the sulfuric acid reaction, either in solution or in SM. The SM procedure is more
convenient and has even greater effectiveness to minimize CO-extractives.It also gen-
erates a smaller volume of reaction extract to facilitate concentration to a smaller vol-
ume for G C examination.
On the other hand, if endrin is present with a larger amount of dieldrin, the use of
the sulfuric acid procedure is not recommended, because the peak height of the dielf-
drin derivative will become significant and interfere with the endrin derivative peak,
due to similarity of retention times of these two derivatives in several commonly used
GLC columns. Under this circumstance, either the CrCl, method or those of Wiencke
and BurkeHoor of Woodham et al."' could be used. However, the CrCI, method is
better (although it can only confirm endrin and not both residues as in the latter two
method^"^^^'^). The reason is that for many types of environmental samples such as
water and certain sediment samples, it is generally necessary to concentrate consider-
ably the reaction extracts prior to GLC examination. Unlike the CrCL method, the
Table 12
METHODS FOR CONFIRMATION O F
DIELDRIN
S~mpleepoxtde
opennlng
reagents (a) to(f)
HSO~/AC' &F
Cl c
15
DIELDRIN
A ~ OAc
O
BF,, possibly
also reagents
(h) & ( I )
Reagents Product Ref.
HBr/HOAc Transbromoacetate" (?) 12Sb

HBr/Ac,O Transbromoacetate" 77
HBr (aq) Transbromohydrin' 77
HCI/Ac20 Chloroacetates (?) 126
ZnCI/HCI Transchlorohydrin 119
BC1,/CI(CHz), OH Transchlorohydrin" 112
Ac20/H,S0, Rearranged diacetatelZ4 14
HZSO, Rearranged p r ~ d u c t s " ~ 14
BF,/diethyl ether Rearranged product^"^ 127, 129b
(see also Volume I,
Chapter 4)
HCIO, Rearranged products 59
Product(s) not identified in original papers. For more de-

tailed discussion and new findings on the complex chem-
istry of dieldrin, see Volume I , Chapter 4, Section VI.
Not originally designed for confirmatory tests but in-
cluded here for historical interest.
considerable contribution of reagent blank together with insufficient sensitivity for

dieldrin greatly limits the applicability of these two methods (Wiencke and Burke and
Woodham et al.112,119) to environmental samples. The various confirmation procedures
for endrin are summarized in Table 11.
C. Dieldrin
Although there are many reactions reported for dieldrin analysis and confirmation
of identity, some as early as the late 1950s,128.129,130
its chemistry is far from well under-
stood. The structures of some of these reaction products and the review of its chemistry
are discussed in Chapter 4. Two key interests in the chemistry of dieldrin are (1) the
cis-opening of the epoxide ring as reported by Chau and Cochrane130and (2) the exten-
sive Wagner-Meerwein rearrangement.12' Understanding its chemistry may assist in the
understanding of its degradation pattern in the environment.
It may be pointed out that, in 1971, Elgar132mentioned several reactions for the
confirmation of dieldrin but without any accompanying data, references, sufficient
experimental conditions, and yield or detector responses of the derivatives. We have
been unable to trace any subsequent papers on the subject. For practical purposes,
these reactions or "procedures" as they were called, are ignored in Table 12.
Dieldrin can be confirmed by sulfuric acid reaction,14 by BFJdiethyl ether,127.129 or
by HC104,124all giving the same major peak by GC analysis. The assignment of the
product by BFJdiethyl ether has been127,129 the noncross-linked dieldrin ketone. In
view of Cookson and Crundwell's work discussed in Chapter 4, Section V1 and the
mechanistic consideration and new findings by ApSimon et al.,125this derivative is
more likely to have at least a cross-linkage.
Although the chemistry of these reactions is interesting, due to the insensitivity of
the procedures using these reactions and multi-products obtained, these reactions have
limited application in the routine confirmation of dieldrin residue in environmental
sample extracts.
The other methods described previously for confirmation of endrin residues,
namely, the HCl/ZnCl2, the BCl,, and H,SO,/AC,O methods can also be used for the
confirmation of dieldrin. These and other methods are summarized in Tables 11 and
12.
The reaction with HBr in anhydrous dioxane was investigated by Parson and
Koore13' in 1966. The product was identified131as transbromohydrin of dieldrin (see
Chapter 4, Section VI). The HBr reaction (probably aqueous HBr [48%]) was men-
tioned, without reference and discussion, by Elgar13' in 1971 to form a cisbromohy-
drin after 100°C, '/2 hr. While cis-opening of dieldrin was observed,lZ6it is under a
different circumstance, and the transisomer is still the major product. Therefore, it is
difficult to visualize a cisisomer as the product, particularly in the presence of water
(HBr acid is 48% HBr in water). In 1973, Maybury and CochranecZ7evaluated eight
reagents, including three using HBr for the confirmation of dieldrin (see Table 12).
The aqueous HBr (48% HBr acid) gave a single product (the bromohydrin) whereas
the HBr/AczO reagent gave to products at 100°C, '/2 hr confirming the earlier report
of Hammence et who also observed that if the same reaction is carried at room
temperature only a single product was obtained. Similarly, HBr in acetic acid (lOO°C,
1/2 hr) gave a single bromoacetate. The major product from the HBr/Ac,O has been
identified16 as transbromoacetate of dieldrin by NMR JJ' coupling and decoupling
experiment, courtesy of Professor J . W. ApSimon. Among these three HBr reagents,
the HBr/Ac,O reaction at room t e m p e r a t ~ r e , ~and ' the HBr/HOAc reaction at 100°C
are similar in sensitivity, giving the same transbromoacetate. The reaction in aqueous
HBr, giving a bromohydrin (most likely with transconfiguration), is less sensitive and
the derivative exhibits a broader peak. Also, its retention time has a tendency to shift
in the same column from time to time. Recently, Musial et a1.lZ6slightly modified the
procedure of Hammence et al." for the confirmation of dieldrin and endrin by substi-
tuting hydrobromic acid with hydrochloric acid in acetic anhydride and carried out
the reaction at 100°C for 45 min instead of 120°C for 30 min. Unfortunately, this
modified method is not compared with the original; therefore, it is not readily known
which one is better. The authors did not mention why they modified the procedure of
Hammence et al. However, due to similarity of reagents and procedure between these
two methods, no significant improvement is expected. Although the products are not
identified, by analogy to previously discussed and similar procedures, one of the deriv-
atives of dieldrin is probably the transchloroacetate. The HCl/ACzO reagent was also
mentioned by Elgar132in 1971 to yield a transchloroacetate at 100°C for 15 min, but
he did not provide any discussion on or reference to this reaction.
The B C l , / 2 - ~ h l o r o e t h a n o land
~ ~ ~ZnCIz/HC1"9 both give the same product. Wiencke
and Burke119identified it as the chlorohydrin. The trans configuration is suggested16
because it was partially converted to dieldrin in the presence of CO-extractiveson
"dirty" G C column. Its trans configuration is now establi~hed,'~' since after reaction
with methanolic KOH, dieldrin is the only conversion product. This product had pre-
viously been postulated as a dieldrin "ketone.""'
According to the evaluation by Maybury and Cochrane"' on eight procedures, the
HBr/HOAc reagentlz8 (lOO°C, 1/2 hr) giving the bromoacetate derivative is the most
sensitive method. One of the procedures (HBr/Ac20 at room temperature) by Hamm-
Volume I 105
Table 13
METHODS FOR T H E CONFIRMATION O F ALDRIN
ADDITION
Expoxidation Ref. Addition Ref.
BY BY
Peracetic acid 133 t-BuOCl/Ac,O 13
Performic acid 13 t-BuOCl/BuOH 13
Perbenzoic acid 13 Chlorination 77
mChlorobenzoic acid 134 Chlorination and bromination 135
Monoperphthalic acid 13 CI(CH,COO),O/HBr 136
Chromic acid 78 Halogenation: solvent effect 137
ence et also gave the same transbromoacetate as the product. If the yield from
both reaction is similar, both methods should give similiar sensitivity. Unfortunately,
the HBr/Ac20 (room temperature) procedure was not e v a 1 ~ a t e d . lThe ~ ~ HBr/Ac20
(100°C127or 120°C77)procedure gave two and hence is less sensitive because
one of the peaks is the transbromoacetate and the height of this peak is decreased at
the expense of the other product peak. Similarly, the HCl/Ac20 (100°C) procedure of
Musial et which generates two peaks, one of which is probably the transchlo-
roacetate as mentioned previously, could have similar sensitivity.
The ZnC12/HCln9 and BC13/2-chloroethanol"2 procedures have identical sensitiv-
ity.lZ7They are only slightly less sensitive than the HBr/HOAc or HBr/AC20 method
discussed above. We found that the bromoacetate derivative is somewhat less prone
to be converted back partially to dieldrin on some well-used GC columns. However,
such conversions were observed to occur, to a greater or less degree, under certain
circumstances (e.g., heavily used columns, or sample CO-extractivesdeposited on glass
insert, presence of traces of acid or alkali). In this regard, these procedures must be
applied with extreme care.
In conclusion, there is no satisfactory method at present for the confirmation of as
low levels of dieldrin as these found in natural water samples and even in some sedi-
ment samples (e.g., Great Lake sediment) because of high reagent blanks and/or in-
sufficient sensitivity.
D. Aldrin
Chemical reactions used for the confirmation of aldrin and summary of confirma-
tion procedures are respectively summarized in Tables 13 and 14.
Halogenation of the unchlorinated double bond constitutes one approach ot its con-
f i ~ r n a t i o n . ' ~S
. ~o' l o ~ a y stated
' ~ ~ that chlorine reacts readily with aldrin to form the
trans-dichloride as the predominant product, while bromine in carbon tetrachloride
forms a mixture of c i s and transdibromide with mp 151 to 152 and 171 to 172"C,
~ ~ is in contrast to the finding of D a ~ i e s , 'who
r e s p e c t i ~ e l y . ' This ~ ~ obtained only
a single transdibromide in high yield. In the related reaction of 1,2,3,4-tetra-
chloro-l ,l , la,5,8,8a-hexahydro-l,4-dimethoxymethano-5,8-methanonapthalenewith
Table 14
SUMMARY OF CHEMICAL CONFIRMATION PROCEDURES FOR ALDRIN
Reaction Known
Aldrin CI,/CHCI, Room temp., 5 min - 77

Aldrin, (heptachlor, DDE) CrO,/HOAc 77"C, 12 min Dieldrin 78
Aldrin Peracetic acid Reflux, 3 hr Dieldrin 133
Aldrin mchloroperbenzoic 43-45°C. 1 % hr Dieldrin 10,134
acid/hexane
Aldrin Monopherphthalic Room temp., 20 min Dieldrin 13
acid/hexane
Aldrin, (helptachlor) t-BuOCI/HOAc 10O0C, 10 min 14
Aldrin Chloroacetic anhy- 56-60°C. 2 hr Not investigated 136
dride/HBr
bromide in chloroform, Mackenziel"O also obtained a single dibromide. Our investiga-

t i ~ n at' ~the~ pesticide residue level indicate that bromination of aldrin at room tem-
perature with bromine in acetic acid, in water, in benzene, or in petroleum ether always
gave two peaks. The major peak has the same retention time on several GLC columns
as that of the transdibromide obtained and isolated by repeating Davies'
e ~ p e r i m e n t . The
' ~ ~ other peak with approximately two times the retention time of the
transdibromide is presumably the cisdibromide. Reaction of aldrin with bromine in
carbon tetrachloride at room temperature also gave two peaks corresponding to the
retention time of the two dibromide peaks mentioned above. In all cases, the trans
dibromide peak predominates, especially when bromination is done in an ionic medium
such as water and acetic acid. Chlorination of aldrin was also utilized by Hammence
et al." for confirmation of its identity. They obtained "a derivative 2.5 times that of
aldrin." In our ~ o r k , ' ~however,~ . ' ~ ~ two products were obtained from similar chlori-
nation reaction using 10% chlorine in chloroform. The main peak (2.8 times the reten-
tion time of aldrin) is identified'37as the transdichloroaldrin whereas the second peak
at 5 times the retention time of aldrin and about one fifth the peak height of the first
peak is presumably 13=thecisdichloro-compound. Chlorination method is almost twice
as sensitive as the bromination method. In fact, up to the time of writing of this man-
uscript, it is the second most sensitive method for the confirmation of aldrin following
the epoxidation procedure of aldrin to dieldrin. The transdichloroaldrin peak will be
interfered with by p,p'-DDD, which is unaffected by the chlorination procedure; how-
ever, if the Reynoldsss or Sans78FlorisilB column cleanup procedure is used (see Table
6 ) p,p'-DDD will be separated from aldrin. If PCBs are present in the sample, they
will elute in the same fraction as aldrin, from the Florisil@ column. The chlorinated
derivative of aldrin will be interfered with by some PCB peaks unless the PCBs are
further separated from aldrin by such procedure as the charcoal/foam-column14' (Ta-
ble 6 ) . Owing to the longer retention time of the transdibromide of aldrin (between
p,p'-DDT and methoxychlor but well separated from them in many GLC columns)
this method has a wider applicability even in the presence of PCBs except in the case
where the aldrin level is very low compared with that of the PCBs.
An alternative procedure13 for identifying aldrin is by reaction with t-butyl hypo-
chlorite (t-BuOC1) either in excess acetic acid or in t-butanol (t-BuOH) to give the
corresponding chloroacetate (in acetic acid) or chloro-t-butyl ether (in t-BuOH). The
number of reaction products in the t-BuOCl/HOAc reaction depends on the t-BuOC1
to acetic acid ratio. Unless the acetic acid is in excess, two products will form; namely,
the chloroacetate and the chloro- t-butyl ether. The chloro-t-butyl ether has a retention
time just slighly longer than that of the dibromide but is quite distinct from that of
methoxychlor. It has the longest retention time among all the aldrin derivatives so far
reported for its identification. Unfortunately, the use of t-BuOCl/HOAc has three
noticeable disadvantages. First, this method produces a product which is less than half
the sensitivity of the chlorination method. Second, it is very difficult to obtain pure
t-BuOC1 and it requires extensive purification before it is applicable to the analysis of
water samples which usually require considerable concentration of the extract due to
low levels of pesticide residues usually present. Third, similar to the case of chlorina-
tion or bromination, but to a lesser degree, it is not uncommon that CO-extractives
from samples such as fish and sediment react with the reagents and give extraneous
peaks which were absent in the blank or in the original extracts. Due to its longer
retention time and despite its relative insensitivity, the t-BuOCl/HOAc method is more
useful in confirming aldrin in the presence of PCBs in environmental samples such as
fish and sediment.
In using these reactions for confirmation of aldrin, particularly the chlorination and
bromination procedure, careful removal of residual traces of the reagents and the
chlorinated solvents must be complete in order to obtain interpretable gas chromato-
grams because these chemicals "poison" the ECD. In an extreme case, a very broad
solvent front lasting for over 1 hr can result. For extracts such as fish which contain
some oily residue, evaporation of the reaction extract to dryness does not give notice-
able loss of the derivatives. For low fat or nonfatty extracts such as water samples,
0.5 m1 of pesticide grade toluene is added to the reaction extract which is then evapo-
rated to 0.5 m1 in 50 to 60°C water bath. If chlorination or bromination is carried
out in a nonhalogenated solvent such as benzene or petroleum ether, the reaction ex-
tract can be washed with either sodium bisulfite, sodium sulfite, or sodium thiosulfate
to remove the chlorine o r bromine.
The most widely investigated reaction for the confirmation of aldrin is probably the
epoxidation procedure by peracids to convert aldrin to dieldrin; this reaction has long
been known as the manufacturing procedure for dieldrin and often mentioned and
q ~ o t e d . ' ~ ~Several
- ' ~ ' authors have reported the use of different peracids such as rn-
chloroperbenzoic acid by Osadchuk and W a n l e ~ s , peracetic
'~~ acid by nor&^,'^^ per-
benzoic and monoperpthalic acid by Chau and Cochrane.l3 Although mchloroperben-
zoic acid will probably be the reagent of choice due to its availability an stability,
problems due to background ECD-GLC interferences are quite commonly encountered
when applied to water samples. This is partly due to the variation in impurities in
commercial mchloroperbenzoic acid and partly due to the concentration step involved
for water sample extracts after derivation. Monoperphthalic acid does not give this
problem, but unfortunately it is comparably less stable and has to be prepared in the
laboratory. As discussed elsewhere, I 3 epoxidation by peracetic acid and performic acid
is less convenient to control and variation of yield of dieldrin is experienced (probably
due to the formation of glycols or esters). Perbenzoic acid, on the other hand, is also
not available commercially and its preparation is more involved than the preparation
of monoperphthalic acid. Epoxidation can be achieved by chromic acid oxidation,"
but this procedure gives a lower yield of dieldrin and is less convenient to work with
than those using peracids. The use of monoperphthalic acid, due to its insolubility in
hexane used in extraction of the product, gives the most convenient procedure.
Another method, developed by Chau and W i l k i n ~ o n at ' ~ the
~ Water Quality Labo-
ratories in Burlington, Canada involves the reaction of aldrin with chloroacetic anhy-
dride and hydrobromic acid. Athough the isolated product, presumably bromochlo-
roacetic anhydride, has been found to have similar ECD sensitivity to that of p,p'-
DDT, these reagents have not yet been applied to routine analysis due to the low yield
(approximately 59%) obtained from reaction of aldrin at the residue scale. Further
investigation is in progress to increase the yield by other experimental conditions.
Aldrin and dieldrin can be confirmed by UV p h o t ~ l y s i s . ~ OThe

- ~ general
~ . ~ ~ disadvan-
tages o f using this approach have been discussed previously. The insufficient sensitivity
of this approach particularly for aldrin and dieldrin and interferences from sample co-
extractives (sediment and fish, for example) make this approach less suitable for envi-
ronmental samples.
E. The Chlordane Group

The two chlordane isomers, c i s and transchlordanes, and heptachlor are some of
the major components of technical chlordane. Heptachlor epoxide is a minor compo-
nent. The chemical composition of technical chlordane is quite complex and is still not
completely established. For general discussion o n the chemistry of 0.c.s including
chlordanes, see References 142 to 147. For specific documents pertaining to chlor-
danes, see References 152 to 161.
The above-mentioned four chlordane components were also used and existed as in-
dividual pesticides until the recent curtailment due to their toxicity and undesirable
effect on the environment (see Chapter 1). However, technical chlordane is still being
used to some extent. Due to their persistence and wide usage previously, the following
discussion will focus on these four compounds.
1. Heptachlor
A variety of methods have been devised for the confirmation of heptachlor residues.
Tables 15 and 16 summarize the chemical reactions used for the confirmation of its
identity. Confirmation procedures for the chlordane group are summarized in Table
17. The presence of a reactive allylic chlorine atom has been the basis for three confir-
matory tests taking advantage of its relatively labile nature. Reaction of heptachlor
with a silver acetate-glacial acetic acid (AgOAc/HOAc) mixture produces l-acetoxy-
chlordene (Table 16), which has a very similar retention time to that of heptachlor
epoxide on an OV-101/OV-210117or a DC-l 1/QF-l c o l ~ m n . However, ~ ~ . ~ ~ under the
same condition, heptachlor epoxide, if not already separated from heptachlor by a
FlorisilB column such as using R e y n o l d ~ ' ~procedure,
."~ is converted to l-acetoxy-3-
chlorochlordene (Table 18), which has a different retention time than l-acetoxychlor-
dene. Alternatively, heptachlor can be reacted with silver carbonate in aqueous ethanol
(AgC03/aq.EtOH) t o obtain l-hydroxychlordene (Table 16). If the latter is already
present in sufficient concentration, it can be analyzed as such. Otherwise, it can be
subsequently converted t o the more ECD responsive silyl ether or the acetate: l-acetox-
ychlordene. Unfortunately, the silyl ether has a retention time identical to aldrin on
commonly used GLC columns such as OV-101/OV-210,117 DC-l l/QF-1,13.14 and SE-
30/AF-1.29 Also, heptachlor and aldrin are eluted in the same fraction by S a n ~ ' , ' ~
mill^','^^ or Reynolds' FlorisilB columns, and also by B e r g ' ~ charcoal
'~~ column and
the charcoal/foam c01umn.l~~ With AgC03/HOAc, heptachlor was the only chlori-
nated pesticides we tested t o undergo modification, and in this regard it is the most
specific chemical reaction observed for any of the compounds we investigated.
A more convenient and sensitive procedure for the confirmation of heptachlor resi-
due identity is the use of chromous chloride for the allylic reductive
dechlorination to chlordene (Table 16). The conversion to chlordene is nearly quanti-
tative and this reaction can be accomplished at 50 to 60°C in less than 0.5 hr with
minimum work-up. Because the procedure is quite simple and the ECD is more respon-
sive to the product than the parent compound, it is by far the most sensitive and quick-
est method for the confirmation of heptachlor identity. As mentioned earlier, endrin
under similar conditions is completely converted to a pentachloro-pentacyclic ketone
(see Table 16) having a different retention time from chlordene. Thus if heptachlor
and endrin are both present in sample and have not been separated by a silica gel or
Table 15
CHEMICAL CONFIRMATION METHODS FOR
HEPTACHLOR
Reaction in Solution
Reagents Product Ref.
Ag2C0,/aq. EtOH followed by I-hydroxychlordene, 1- 149

acetylation or silylation acetoxy chlordene, silyl
derivative
I-Acetoxychlordene 149
t-Butyl acetate derivative 14
l-Hydroxychlordene 13
Chlordene 15,17,150
Reaction o n Solid Matrix
t-BuOK/AL03 l-Hydroxychlordene 60
KOH/AI,O, l -Hydroxychlordene 60
Reaction TLC l-Hydroxychlordene 67
H,SO,/AI,O, I-Phenylchlordene (prod- 63
uct identified later)
H2SO4/HZO/Al20, l-Hydroxychlordene 65
FlorisilO column (see Table 6), they can be confirmed simultaneously by chromous
chloride reduction in acidic medium. As discussed previously (see section on endrin),
the acidity of the chromous chloride solution is important for clean reaction. In con-
trast, reductive dechlorination of heptachlor is unaffected by the pH of the media.
The use of chromous chloride solution for confirming heptachlor identity, however,
has a major drawback in that the chlordene formed has a very short retention time on
OV-101/OV-210, DC-1 1/QF-l , and similar GLC column retention time (identical or
similar to lindane, which disappear under the same reduction condition). In many
"dirty" samples, CO-extractive peaks usually appear in that area although it is not
uncommon that these CO-extractivepeaks are decreased or eliminated after chromous
chloride reduction.
Two other methods for the confirmation of heptachlor involve addition to the ster-
ically hindered and inert (due to the adjacent chlorine atom) double bond (see Table
16). Normally chlorine does not add to the heptachlor unless an initiator such as anti-
mony pentachaloride is present. Also heptachlor is resistant to epoxidation by pera-
cids. However, additionI4 and e p o ~ i d a t i o n ' ~can , ' ~ ~readily be achieved chemically by
~-BuOC~/HOAC and
' ~ chromic oxide ~ x i d a t i o n , ' ~ .respectively.
'~~ Analogous to the
reaction of aldrin with t-BuOCl/HOAc mentioned previously, the reaction product is
influenced by t-BuOC1 to acetic acid ratio. In excess acetic acid, the t-BuOCl/HOAc
reaction on heptachlor produced one peak which corresponds to the chloroacetate of
h e p t a ~ h l o r Otherwise
.~ two peaks corresponding to the chloroacetate and the chloro-
t-butyl ether are observed. This situation is similar to that observed for the reaction
of aldrin with these reagents. Since all these peaks have a different retention time from
each other, aldrin and heptachlor can be confirmed simultaneously by this method.
The ECD is about twice as sensitive towards the chloroacetate of heptachlor as com-
pared with aldrin chloroacetate.
Recently, Chau and Terry63 reported another method for the confirmation of hep-
tachlor using SM derivation technique. This procedure involves the reaction at 100°C
of heptachlor or a concentrated sample extract in a micro reaction column, packed
with alumina and sulfuric acid in the ratio 50 g to 10 m l . After 1 hr at 100°C, hepta-
Table 16
REACTIONS USED IN THE CONFIRMATION O F HEPTACHLOR
HEPTACHLOR \- I-HYDROXY- CHLORDENE
I-ACETOXY- SlLYL ETHER

CHLORDENE
HEPTACHLOR
CHLORDENE
chlor is completely converted in approximately 80% yield to a derivative, the structure

of which has now been determined as l - p h e n y l c h l ~ r d e n e '(Table
~~ 16). The ECD has
similar response toward this derivative as DDT. This derivative and the chloro-acetate
derivative mentioned above have similar retention times and are eluted slightly after
DDT on an OV-101/OV-210,"' DC-l 1/QF-1, or similar column. Due to their longer
retention time and the derivative peaks being removed from common O.C.pesticides,
the SM and the t-BuOCl/HOAc procedure are generally preferred. For samples such
as water containing low level of heptachlor and CO-extractives,the chromous chloride
reduction method is preferred. The t-BuOC1 addition method, as discussed earlier,
suffers the disadvantage that the reagent is difficult to purify and is quite unstable.
The interference peaks will be intensified if the reaction extract is concentrated to a
low volume, a step necessary for most water samples. The SM procedure is much
simpler to control and execute as compared to the t-BuOCl/HOAc method. Also many
Table 17
SUMMARY OF CHEMICAL CONFIRMATION PROCEDURES FOR THE CHLORDANE
GROUP
Reaction Known
Heptachlor epoxide 120°C. 30 min or room

temp., 30 min
Heptachlor, (aldrin, DDE) CrO,/HOAc 77"C, 12 min
Heptachlor AgOAc/HOAc Reflux, 30 min
Heptachlor Ag2C03/50% aq. EtOH, Reflux, 45 min
then silylation or acetyla-
tion
Heptachlor, its epoxide, t-BuOK/ t-BuOH, then si- Reflux, 30 min 2- and 3-chlorochlordene 13,14
c i s and transchlordane lylation or acetylation Nonachlor
(DDT + DDD isomers)
Heptachlor (and endrin) CrCl, aq. acidic sd. 55-6O0C, 45 min Chlordene 17
Heptachlor, its epoxide, cis AI2O3/t-BuOK 70-75"C, 1 hr or room 2- and 3-chlorochlordene 60
and transchlordane A1,03/KOH temp. overnight
(DDT + DDD isomers,
perthane methoxychlor)
Heptachlor, endosulfans, - 63
endrin
Heptachlor, endrin - 65
Heptachlor, its epoxide, en- Room temp., 30 min Not fully investigated 126
drin, dieldrin, endosulfans
Heptachlor, its epoxide UV in petroleum ether 30 min Heptachlor + octachlor 148
(traces of MeOH) epoxide derivatives on
some column; UV deriv-
ative of heptachlor epox-
ide and parent epoxide
separable o n OV-101
column
Table 18
REACTIONS USED IN T H E CONFIRMATION O F HEPTACHLOR EPOXIDE
OH
Cl 1- HYDROXY-
HEPTACHLOR 3-CHLOROCHLORDENE
OAc
Cl
Cl
SlLYL ETHER 1- ACETOXY-
3-CHLOROCHLORDENE
of the CO-extractivespresent in the sample extract are destroyed giving a clean chro-
matogram. Therefore, the SM procedure is the preferred one. As will be discussed
later, both a- and /?-endosulfans are qantitatively reacted by the same SM procedure
to an ether (Table 22) with a different retention time from the the heptachlor derivative
(l-phenyl chlordene). Thus if these pesticides are not separated during the column
cleanup procedures, they can be simultaneously confirmed by the same SM derivation
method. Worth mentioning is the fact that all the above chemical derivation methods
for confirmation of heptachlor identity are generally not applicable if PCBs are present
due to the interference of the PCB peaks with the derivatives.
Since heptachlor and PCBs are CO-elutedfrom most cleanup and fractionation col-
umns described in multi-residue procedures in many analytical methods manuals, the
column eluate needs t o be further treated by coconut charcoal column as described by
Berg et aI.ls3 or the silicic acid/Celite@ column as described by Armor and B ~ r k e ' ~ ~
to separate heptachlor from PCBs before performing either one of those chemical
derivation experiments so far mentioned. This approach is rather time consuming.
Also, difficulties have been encountered in application of these methods to separate
small amounts of some 0.c.s such as heptachlor from the usually much larger amounts
of PCBs encountered in many environmental samples. These difficulties result mainly
from the incomplete separation of PCBs from heptachlor by these methods. This sit-
uation is further complicated by the variance in activity of different batches of adsor-
bent and by the unpredictable effect of CO-extractivessuch as lipids and oils on the
separation characteristics of these adsorbents for these two classes of compounds with
such close column eluting characteristics. This is particularly true when the usually
small amounts of heptachlor are encountered in the presence of a 100-fold greater
quantities of PCBs.
To overcome this problem, a simple procedure has been devised65by taking advan-
tage of the characteristics of SM derivation technique. By this newly devised proce-
d ~ r ederivatization
, ~ ~ of heptachlor to l-hydroxychlordene (90% yield) and subsequent
separation form the unreacted PCBs is carried out in one step on an alumina-sulfuric
acid column. Because of the diversity in polarities of l-hydroxychlordene and the un-
reacted PCBs, they are distinctly separated. Also, due to the presence of sulfuric acid
and somewhat drastic conditions (1.5 hr at 95" to 100°C for reaction) used in this
derivatization the chromatograms of the column eluants (PCBs and l-hydroxychlor-
dene fractions) after reaction are often much "cleaner" than those before reaction
since many CO-extractivesare destroyed and/or adsorbed onto the SM after reaction.
This not only enables confirmation and identification of heptachlor at a considerably
lower level but eliminates CO-extractivesthat could be interpreted as some of the PCBs
peaks. If required the l-hydroxychlordene can be further ~ i l y l a t e d 'or
~ ~a ~ e t y l a t e d ' ~ ~
to increase detection sensitivity.
Alternatively, a charcoal/foam c01umn'~' can be used to separate PCBs from hep-
tachlor (Table 6). Unlike other adsorbants such as FlorisilB and silicic acid, the char-
coal/foam material is less subject to variation due to laboratory or manufacture
source. Moreover, since two solvents of different polarities (cyclohexane and benzene)
are used for the respective elution of heptachlor and PCBs, the procedure is more
rugged than those using the same solvent to obtain two fractions. Nevertheless, in the
presence of large quantity of PCBs in relation to heptachlor, the above-discussed SM
technique is still the most reliable procedure for the confirmation of trace amount of
heptachlor.
2. Heptachlor Epoxide and Chlordane Isomers

At the time of writing this manuscript, there are only 2 practical chemical and one
photochemical derivation methods for the confirmtion of heptachlor epoxide (see Ta-
ble 17), an alkaline treatment using potassium t-butoxide/t-butanol (t-BuOK/t-BuOH)
or sodium methoxide/methanol (NaOMe/MeOH) followed by silylation or acetylation
and the method of Hammence et using HBr/Ac20 or its modified procedurelZ6
using HC1/Ac20.
The HBr/Ac,O or HCl/Ac20 procedures gives considerable reagent background in-
terferences and is not suitable for low level (sub-ppb) confirmation particularly when
concentration of the reaction extract to 0.5 to 1 m1 is required. Moreover, these meth-
ods are narrow in scope in that they are not applicable to isomers of DDT and chlor-
dane. O n the other hand, the t-BuOK/ t-BuOH procedure can simultaneously confirm
several pesticides, particularly those in the same eluant (see Table 6) from the com-
monly used FlorisilB column.
Between NaOMe and t-BuOK, the use of t-BuOH/t-BuOK is preferred because of
shorter reaction time and most important of all, because this reagent can simulta-
neously confirm heptachlor, heptachlor epoxide, trans and cischlordane and DDT-
type compounds. Thus, trans and cischlordane are dehydrochlorinated to 3-chloroch-
lordene and 2-chlorochlordene, respectively (Table 19), whereas heptachlor is con-
verted to l-hydroxy-chlordene (Table 16), and heptachlor epoxide to l-hydroxy-3-chlo-
rochlordene (Table 18). To enhance ECD response, the hydroxy compounds can be
subsequently silylated or acetylated to the respective silyl ethers or acetates; 3-chlo-
rochlordene and 2-chlorochlordene are not affected by the silylation or acetylation
procedures. The DDT-type compounds are converted to the respective olefins which
are also not affected by subsequent silylation or acetylation procedures. Reactions of
the above compounds can be performed in ~ o l u t i o n ' ~or
. ' ~on SM.60The general advan-
tages of using SM derivation technique have been discussed previously in this review.
In particular, using this approach offers three other advantages. The first is that the
eluants from the reaction micro-column containing derivatives to be analyzed by GLC
are free from traces of alkaline, whereas if the reaction is carried in solution, some
alkali could also be extracted into the extraction solvent. Traces of alkali accumulated
and deposited on the glass insert or the head of the column through routine injection
of reaction extracts can partially modify pesticide residues in a manner similar to re-
action gas chromatography using an alkaline precolumn in the injection port. This
happens when reaction extracts from derivation carried out in solution were not suffi-
ciently washed with water to remove the alkalinic materials and the GLC column has
Table 19
REACTIONS USED IN T H E CONFIRMATION O F CHLORDANES
CIS-CHLORDANE TRANS -CHLORDANE uH
been used continously for a period of time to analyze these reaction extracts. To avoid
this problem, the reaction extracts must be thoroughly washed with water and dried
over anhydrous sodium sulfate before GLC analysis. These procedures are time con-
suming and also induce mechanical loss. SM derivation technique eliminates these
problems.
The second advantage is that using the SM dehydrochlorination procedure the pres-
ence of nonachlor, one of the major constituents in technical chlordane,16' does not
interfere with the confirmation of cischlordane, another major constituent in technical
chlordane. This is in contrast with the dehydrochlorination in solution. Nonachlor and
cischlordane have identical or almost identical retention times in many GLC columns
such as an OV-101/OV-210, DC-1 l /QFl and 3% SE-30 column. Their respective de-
hydrochlorinated products: 1,2-dichlorochlordene167and 3 - c h l o r o ~ h l o r d e n ealso
~~~~
have a very close or identical retention time in these columns and appear as a single
peak if present together.60 Dehydrochlorination of nonachlor and cischlordene in so-
lution yields these two GLC indistinguishable products in many GLC columns. In con-
trast, dehydrochlorination on SM does not pose this problem because nonachlor pres-
ent is up to 1 pg after reaction on SM,60 does not give any significant derivative peak
in the gas chromatogram although it was completely reacted. On the other hand, c i s
chlordane gives 3-chlorochlordane in good yield.60 Thus, cischlordane can be con-
firmed in the presence up to 1 pg of nonachlor by the SM derivation procedure.
Lastly, similiar to the case of other SM procedures such as those for endrin6" and
h e p t a ~ h l o r ,using
~ ~ . ~Al,O,/H,SO,
~ always results in a much "cleaner" chromatogram
due t o destruction of CO-extractivesby the strong alkaline system (t-BuOK), high re-
action temperature, or hold up of CO-extractivesin the reaction column.
Unfortunately, there is one major disadvantage in using this SM (alumina/t-BuOK)
derivation procedure. Heptachlor and its epoxide, although completely reacted, do not
give any significant derivative peaks and none of the expected hydroxy derivatives was
detected. If alumina/KOH is used, the expected hydroxy compounds are formed.
However, this conversion requires 2 to 3 hr for complete conversion and the yield of
l-hydroxychlordene was 30% lower than that from the same t-BuOK/ t-BuOH reaction
carried out in solution. However, if during the column cleanup, a procedure such as
that reported by R e y n o l d ~ heptachlor
,~~ is separated from heptachlor epoxide, c i s and
transchlordane, DDT, DDD, and methoxychlor, the confirmation of heptachlor can
be separately performed by one of the methods (Table 16) mentioned previously. The
various confirmation procedures for the chlordane group are summarized in Table 17.
Table 20
METHODS FOR T H E CONFIRMATION O F
ENDOSULFANS
Reaction in Solution
Reagents Products Ref.
Sulfite cleavage
Ac,O/H,SO, Diacetate 11
LiAIH,/THF Diol disilyl ether
+ 11
Diol -, diacetate
HCI/Ac20 Possibly diacetate or chloroacetate 12
Multi-sites Rx
KOH/ROH Cyclic ether 168
Reaction in Solid Matrix
Sulfite cleavage
AclO/H2SO,/AI1O3 Diacetate
H,SO,/AI,O, Cyclic ether
F. Miscellaneous 0.C.s
l. Endosulfans (a-and p-Isomers)
The terms a- and /3-endosulfan are synonymous with endosulfan a, A, I, and Thio-
d o n 8 A or I. p-Endosulfan is synonymous with endosulfan b, B, 11, and Thiodan8
B or 11. ThiodanB is the registered trademark of Canadian Hoechst Ltd. for pesticide
formulations containing endosulfans.
So far, there appear to be four chemical reactions (Tables 20 to 22) utilized for the
confirmation of endosulfans by the derivation-gas chromatographic technique,
namely, (1) a single-step reaction using acetic anhydride/sulfuric acid (AC20/H2S0,)
mixture either in solution" or on SM61 (alumina impregnated with this reagent) to
convert both isomers of endosulfan to the same diacetate; (2) a two-step reaction,"
consisting of a reduction step with lithium aluminum hydride (LAH) to the diol fol-
lowed by subsequent silylation or acetylation; (3) a one-step base-catalyzed intramole-
cular reaction,l6" carried out in solution to convert both endosulfans to an ethoxy or
methoxy internal ether; and (4) a sensitive and quick SM derivation p r o ~ e d u r e ~to ',~~
convert both endosulfan isomers to the same ether using a micro-reaction column im-
pregnated with alumina/sulfuric acid. The reaction pathways for these reactions are
summarized in Table 22.
Recently, Musial et reported a slightly modified procedure of Hammence et
(HC1 instead of HBr77in A c 2 0 and retaining all the original77experimental con-
ditions) and extended the method to the confirmation of endosulfan isomers. These
obtained a product at a relative (to aldrin) retention time of 2.54 from a-
endosulfan and 2.51 from 0-endosulfan. Both products probably were identical or c i s
and transisomers.
Unfortunately, these authors did not attempt to identify the products and the rela-
tive retention times of their derivatives as well as those of the parent compounds and
other pesticides are dissimilar to those reported p r e v i o u ~ l y . ~Based
' ~ ' ~ on the chemistry
of endosulfans and the nature of their derivatization reagent, the product could be
either the diacetate1'.16 or a chloroacetate. The reaction conditions of all the confir-
mation procedures discussed are summarized in Table 18.
The LAH reduction method involves two reactions: first the endosulfans are con-
verted to the same diol which has been observed as one of the metabolic pro-
dUCtS;169-175second this diol is reacted with silylating or acetylating re-
Table 21
SUMMARY O F CHEMICAL CONFIRMATION PROCEDURES FOR
ENDOSULFANS
Reaction Known
a- and 0-endosulfan LiAIH,/THF, then silyla- 60°C. 1 hr - 11

tion or acetylation
Ac20/HOAc/trace of 100°C, % hr - 11
HSO4
2% KOH in EtOH Reflux, 12 min Not investigated 168
A1203/Ac20/H2S04(50g/ 100-c5"C, 2 hr - 61
10 m!/5 m!)
AI,O,/H,SO, (50g/10 m l ) 95°C. 1 hr - 62
a- and I]-endosulfan, A1203/H2S0,(50 g/10 m!) 100°C, 1 !h hr - 63
(heptachlor)
a- and 0-endosulfan 0.5 m! HCI/Ac,O Room temp., 30 min Not investigated 126
(heptachlor epoxide,
endrin, dieldrin)
agent".'72 t o the respective disilyl ether or diacetate. The conversion of diol to a ECD
more responsive compound using selenium dioxide for detecting this diol has also been
reported.17' Although this LAH reduction procedure is a relatively longer procedure,
it offers an advantage in that this very strong reducing agent destroys most, if not all,
of the CO-extractives, even in a straight sample extract of fish, water, and sediment.
Most of the common pesticides are destroyed completely or peak heights attributed to
them decrease drastically, probably because of conversion to ECD insensitive com-
pounds. Thus the chromatogram of the straight sample extract, after LAH treatment
and subsequent acetylation or silylation, is quite "clean". PCBs AroclorB 1260 and
1254, after LAH reductions at 10 pg in 5 m! in 5 m! level, gave only three or four
peaks less than 10% scale of the recorder response. Therefore, the LAH method is a
quick screening method for endolsulfans even in the presence of up to 10 pg of the
two PCB mixtures without cleanup and prior separation.
The one-step acetylation in solution" or on SM61is simpler, but some type of sample
cleanup is required before this reaction is performed. Particularly in the presence of
PCBs, some pre-column separation such as using a FlorisilO column (Table 6 ) is nec-
essary. These acetylation procedures are very similar in sensitivity. The one performed
on SM has certain advantages such as the simplicity of the procedure: no neutralization
and extraction of reaction mixture are required; and minimum mechanical loss due to
the design of the procedure. It also causes further cleanup of the extracts during a
reaction resulting from physical retention of CO-extractiveson the reaction column,
chemical destruction of CO-extractivesby sulfuric acid, and high temperature (lOO°C)
used in the procedure. Of all the SM procedures so far d e ~ e l o p e d in ~ ~these
- ~ ~labora-
tories, the SM method6' used for acetylation of endosulfans, i.e., alumina impregnated
with acetic anhydride and sulfuric acid is the least rugged because it requires extra
careful preparation and storage. This is due to the slow hydrolysis of acetic anhydride
to acetic acid by sulfuric acid, thus eventually rendering the solid material useless for
this purpose. Also, care must be taken to protect the SM from exposure to moisture
which drastically hastens the above-mentioned hydrolysis. Lastly, this SM can only be
kept at room temperature for 1 or 2 months depending on atmospheric moisture and
extent of exposure before the yield of the endosulfan acetate starts to decrease.
In contrast, another SM62prepared from alumina impregnated with sulfuric acid
alone is not only easier to work with, but also has a long shelf life. By this reaction,
both isomers form an ether, identical to that reported by Forman et a1.176-178 It has
also been found as a m e t a b ~ l i t e ' ~ ' ~ 'of
~~ -~~
both ~ . ' ~ ~ isomers.
endosulfan
Volume I 117
Table 22
REACTIONS USED IN THE CONFIRMATION OF ENDOSULFANS
ENDOSULFAN ETHER
1 18 Analysis o f Pesticides in Water
Among these methods, the one using base-catalyzed reaction16' is the least sensitive,
probably due to the relative insensitivity of the ECD towards the alkoxyl ethers and/
or the low yield of these derivatives from this procedure. The sensitivity of the HC1/
A c 2 0 methodtz6is not specifically mentioned'26 for endosulfans except that a detection
limit in the range of 0.5 to 1.0 ppb for the 5 0.c.s tested was mentioned. Due to the
sketchy and incomplete data presented, it is also difficult to obtain some idea on the
yield of the derivatives or their ECD responses in relation to the parent pesticides.
However, in considering the higher reagent blank, the inability of the reaction to de-
stroy CO-extractives,and the possible nature of the derivative (diacetate or chloroace-
tate), the HC1/Ac20 procedure would be expected to have similar sensitivity to that
of the A c 2 0 / H 2 S 0 4reaction" in solution. The LAH procedurell and the Acz0/H2S04
reaction in SM6' give cleaner reaction extract (reduction of CO-extractivesin both cases
and cleaner reagent blank in the case of LAH). The most sensitive and convenient
confirmation procedure for endosulfans is alumina/H2S04SM procedure63to generate
the extremely ECD-responsive endosulfan ether.
Because of the high yield of endosulfan ether (84%) obtained by this method and
also because of the considerably higher ECD responses as compared to the parent
compounds, this method is by far the most sensitive published to date. For the same
amount of endosulfan reacted, an ether peak obtained from the present procedure was
approximately three t o four times higher than that of the diacetate derivative obtained
by acetylation. In turn, the diacetate peak is approximately three times higher than
the internal ether obtained from the procedure of Greve and Wit.'=' This SM derivation
procedure is almost as effective as the LAH reaction in removing CO-extractivesin
"dirty" samples such as fish and sediment. Because of its simplicity this procedure is
also a promising rapid screening method for endosulfans without cleanup.
Based o n the above discussion, the sensitivity of these methods, in decreasing order,
is as follows:
Al,O,/H, > LAH + ~ i l y l a t i o n '>

~ LAH + acetylation" > H, SO,/Ac, 0 in SM6' > H2S04/Ac10
In consideration of procedural simplicity alone, naturally a one-step reaction is sim-

pler than a two-step reaction. Obviously, if other considerations such as sensitivity and
reproducibility of a method, reagent blank and side products, and ability of a proce-
dure t o destroy sample coextractive (see Section V) are equal, then a simpler procedure
is desired. However, it is rare t o have a procedure having all these considerations on
the favorable side; for example, a simpler procedure may suffer from high reagent
blank and/or low sensitivity whereas a more lengthy procedure may have good sensi-
tivity and low reagent blank.
In the present case for endosulfans, it was pointed out by Greeve and Wit'68 in 1971
that the simplicity of their method (one-step KOH/ROH reaction) "is an advantage
over the method used by Chau (1969)" in which reduction with LAH and subsequent
silylation have to be carried out prior to gas chromatographic analysis."168 While this
statement is true if and only if simplicity of procedure is considered, as discussed
above, other factors must be taken into account to obtain a realistic evaluation. In
this case, the disadvantage of the lack of sensitivity of the KOH/ROHI6' outweighs
its advantage in simplicity. Moreover, these authors16' ignored the other procedure (a
one-step H z S 0 4 / A c z 0reaction to form the diacetate) also reported in the same paper
in 1969.11 There is no advantage as far as simplicity of procedure between the H2SO.d
A c 2 0 method1' and the KOH/ROH method." In fact, as discussed previously, this
one-step HZSO4/Ac20reaction and, for that matter, the LAH two-step reaction have
the advantage of being much more sensitive than the KOH/ROH method.
When one is t o discuss the pros and cons of a method, it is the obligation of the
author(s) as well as good scientific practice to be aware of and attempt to consider as
many key criteria as possible t o avoid presenting a misleading picture to the readers.
Another example of misinformation is that of Musial et who stated that "pre-
viously reported methods for derivatization and confirmation of endosulfan isomers
(Chau and Terry 1972,63Chau 197262)tend to be lengthy because the appropriate solid
columns have to be activated. The method reported here (HC1/Ac20 in solution) is
more rapid and aH insecticides examined can be confirmed using a single reagent."'26
While the HC1/Ac20 reagent can confirm five pesticides (endrin, heptachlor epoxide,
dieldrin, and two endosulfan isomers) and hence is more convenient if the pesticides
are not already pre-separated before reaction by a FlorisilB column, due to the similar
retention time of several derivative peaks (see Table 1 of Reference 126) this could
cause confusion in interpretation. It is also not known whether the presence of other
pesticides could interfere. Besides, for chemical derivatization the authors126used three
different FlorisilB column eluants: ether/hexane - 5/95, 15/85, 50/50 for reaction
(see Reference 69 and Table 6). In other words, for the confirmation of the five insec-
ticides examined, three separate reactions have to be carried out because heptachlor
epoxide is in the 5/95 ether-hexane fraction; a-endosulfan, endrin, and dieldrin are in
the 15/85 ether-hexane fraction; and p-endosulfan is in the last fraction.69 The only
advantage of this method'26 is that it can conveniently confirm endrin, dieldrin, an a-
endosulfan, which are eluted in the same fraction from a FlorisilB column.
As discussed so many times, the general advantage of SM procedures is the minimum
mechanical steps (no extraction and neutralization of extract required) and also its
reproducibility due to being less subjective to laboratory variation as procedures are
carried out in solution. In particular, the ether generating SM procedure for endosul-
fans62is so simple and straightforward that a few dozen reactions can be easily carried
out simultaneously even by less experienced personnel. It would be less convenient t o
d o so in the case of reactions carried out in solution since they require several extrac-
tions. Also, this SM procedure can confirm endosulfan and heptachlor simultaneously.
The disadvantage is that it cannot be used to confirm dieldrin as in the procedure of
Musial et However, unlike the less desirable H2S04/Ac20/A1203SM,61 the
A1203/H2S04SM,62once prepared and kept tightly covered in a jar in a desiccator,
can be used for several months without deterioration. In the procedure by Musial et
and for that matter in other it is more lengthy to carry out the
reaction at 100°C1'~'26or at reflux t e m p e r a t ~ r e , 'cool,
~ ~ and extract the derivative from
an or a l k a l i n i ~medium.
l~~ Due to the strong acidic or alkaline medium used,
it is required to neutralize, wash, and dry the reaction extract before GLC examination;
otherwise, traces o f acid or alkali accumulated upon repeat injections on the injection
port or deposited onto the inlet end of the GLC column can severely affect GLC per-
formance. As outlined in the procedure126the insufficient treatment (lack of washing
and drying step) of the reaction extract after washing with saturated sodium carbonate
solution will affect GLC performance upon repeated usage as experienced in the pro-
cedures of DDT dehydrochlorination when there is insufficient provision to remove
traces of alkali carried over into the organic extracts.
In addition to chemical derivatization procedures discussed above, a- and /3-endo-
sulfans can be confirmed by UV photolysis on thin-filmlT2or in hexane solution'80
after trapping in a TeflonB tube according to K a ~ f m a n ' s technique. ~ ~ , ~ ~ As discussed
earlier for other O.C. pesticides, the UV photolysis technique is not sensitive enough
for most environmental application and depending on sample type and location it also
has a greater tendency for interferences from sample CO-extractives.The detection limit
for endosulfan is around 25 to 75 ng.Ie0
2. Mirex
The significance and analysis of mirex and its related compound, kepone, are dis-
cussed in Afghan.330Mirex and kepone can be extracted and the extract cleaned up by
the commonly used multi-residue methods mentioned in standard methods manuals
such as PAM and WQB AMM68.69(see also discussion in Reference 180). Being a
nonpolar compound, mirex is CO-elutedwith PCBs during column chromatography;
for example, in the FlorisilB column PCBs and mirex are co-
eluted with hexane or petroleum ether in the first fraction (see Table 6). Since these
solvents are already the least polar solvents in the eluotropic series (see Chapter 2),
the only alternatives in the attempt to separate mirex from PCBs are either to use a
longer column or to split the hexane or petroleum ether elution into two fractions. It
is possible to achieve this separation under rigidly controlled laboratory conditions,
but this is difficult to achieve and maintain in normal routine operation. The variation
of activities of FlorisilB and silicic acid from batch to batch is well known. Even if
their adsorption activity is rigorously controlled in the analyst's laboratory, the varia-
tion of CO-extractivesfrom sample to sample can make this critical separation nonre-
producible. (For a more detailed discussion see References 141, 181, and 182.) How-
ever, using a charcoal/polyurethane foam micro-column141 in conjunction with a
FlorisilB c o l ~ m n (see ~ ~ Table
. ~ ~ 6) both mirex and PCBs can be quantitatively and
effectively separated into two distinct fractions. Since two solvents of different polarity
are used for elution, this separation is more rugged than using the same solvent for
separate fractions as in the case of using F l ~ r i s i l Bon~ silicic
~ ~ ~ acid
~ ~ column. The
overall schematic for the analysis of mirex, PCBs, and 16 0.c.s is summarized in Table
6. Mirex can then be analyzed and its identity confirmed without PCBs interference
(However, see later discussion on PCB interference.)
Currently, there are three approaches for the confirmation of mirex by chemical
reactions. All these approaches are designed to be used in conjunction with multi-resi-
due methodology for 0.c.s and PCBs. (As mentioned previously, PCBs and mirex will
be in the hexane or petroleum ether fraction if the FlorisilB or silicic acid column is
used).
The first two approaches are based on the well-known chemical procedure^^^^-'^^
originally developed for PCB analysis in the 1960s and 1970s. They are the perchlori-
nation192.181 and n i t r a t i ~ n ' ~ ~procedures.
.'~' Under the conditions used in these proce-
dures, mirex is not reacted, but PCBs are either transformed to decachlorobiphenyl in
the perchlorination procedure or to the nitrated PCBs in the nitration procedure. These
PCB derivatives are less volatile and have a quite different retention time than the
unreacted mirex. Being more polar, they are also easily separated from mirex by col-
umn chromatography; thus mirex can be quantitated. The presence of the mirex peak
before and after chemical reaction is inferred as substantiation of its identity. These
procedures can only be considered as indirect, semiconfirmatory techniques because
mirex is not reacted.
At the time of writing this manuscript, the only published direct chemical confir-
mation of mirex is the use o f chromous chloride (CrC1,) reductive dechlorination re-
aCtion.182.194.195The cyclodiene pesticides and mirex have the same gem-dichloro groups
and mirex possesses comparatively less reactive bridge-head chlorines than the cyclo-
diene pesticides. Based on considerable past experiences with CrC1, reduction of cyclo-
diene pesticides (see previous discussion), the reaction of mirex with CrCL is therefore
a logical choice for the investigation of a confirmatory procedure for its identity.
Using different ratios of aqueous CrC1, solution and acetone and different reaction
conditions, three procedures for the reduction of mirex were d e v e l ~ p e d ' ~as
~ ,confir-
'~~
matory tests. Each procedure gave a major peak with different retention times on a
3% OV-101 GLC column under common operational conditions used for O.C. analysis.
The structures of these products are not known yet, but based on GLC retention times
on several single and mixed phase GLC columns, the major peak obtained from each
procedure is not the photomirex (8-monohydro derivative of mirex) or the 2,8-dihydro
derivative of mirex synthesized in our l a b ~ r a t o r y . ' ~ ~ , ' ~ '
In early 1979, Lusby and reported that another "simple chromous chloride
reduction, following the techniques developed (by Chau et a1.),.'2.'5.'9,122.123.151 yielded
products from mirex which are eluted in the area of chromatogram free from PCB
component^"'^^ (ArochlorB 1260). Unlike the procedures (CrC1, in acetone at room
temperature and at 60°C) developed by Chau et a1.'82,195 for the confirmation of mirex
in 1978, Lusby and Hill'94 used a 2: 1 dimethyformamide/chromous chloride solution
and stronger reducing conditions (100°C for 45 min). Dimethylf~rrnamide'~~-~~ is an-
other common solvent used for CrC1, reaction.
The relative retention time (to mirex) of the product from Lusby and Hill's proce-
d ~ r e is' ~estimated
~ to be 0.15 by measuring the chromatographic tracings in their
paper, whereas the dominant peak in one of the procedures (CrCl, in acetone at 60°C
overnight) reported by Chau et al.'82,195 has a relative retention time of 0.14. This peak
(labeled as Peak "needs more energy (a higher reaction temperature) to predomi-
nate. It becomes a minor peak when the reaction is carried out at room temperature
even after 4 days of reaction."la2 At 60°C in 12 to 15 hr however, it becomes a major
peak and mirex is completely reacted. At room temperature, there are only 2 major
peaks and the acetone/CrC12 ratio controls which peak predominates. At 60°C, the
effect of different ratios of acetone/CrC12 had a less pronounced influence on the yield
of the three product peaks than at room temperature. In other words, at 60°C, all
the acetone/CrCl, ratios investigatedta2gave Peak I as the major peak. Thus, any one
of these three peaks, probably from the more stable intermediates of dechlorinated
mirexes can be obtained as the major peak at the expense of the others. Further deg-
radation (dechlorination) of Peak I requires even more drastic condition, namely, the
chromous chloride-ethylenediame complex198~200-202 as applied p r e v i o ~ s l yto
' ~the
~ ~de-
~~
chlorination of resistant compounds such as aldrin,15.203 dieldrin,'s,204and a few hep-
tachlor derivatives.208However, the products have poor ECD response for most prac-
tical residue application due to excessive dechlorination.
The procedure'82 of dechlorinating mirex using 1 m1 CrCl, solution in 5 m1 acetone
at 55 to 60°C overnight (12 to 15 hr) to obtain Peak I is the most sensitive procedure
among the three procedures described by Chau et al.'', It can detect 1.1 times the
amount of mirex standard (taken as 1.0), whereas procedures to generate Peaks I1 or
I11 can detect 0.7 and 0.5 times, respectively. On the other hand, the procedure of
Lusby and which probably also generates Peak I as the predominant product,
can detect 0.69 times the amount of mirex, taken as 1.0. This calculation is based on
the peak heights of mirex and its product estimated from chromatographic tracing in
their p ~ b l i c a t i o n . 'This
~ ~ lower sensitivity is due to the more drastic reduction condi-
tion to cause further dechlorination of mirex. It is similar to the situation of using
CrC1,-en complex (CrC1,-ethylenediamine complex) in the reduction of mirex.lU2Thus,
more of the poorer (less chlorine atoms) ECD-responsive products are probably pro-
duced at the expense of Peak I.
Since Peak I is in the area where ArochlorB 1260 peaks are insignificant, mirex can
be confirmed by CrC1, reduction by either p r ~ c e d u r e . ' ~ ~ However,
.'~~ in many fish,
sea gulls, and lake sediment extracts, lower chlorinated PCB from lower
ArochlorsB are present in this area thus causing interference in the confirmation of
mirex by the CrC1, methods. In this case, before reduction of mirex, it can be separated
from PCBs by a mini-charcoal/foam column (Table 6) as described by Chau and Bab-
jak.141
The UV photolysis of mirex in hydrocarbon205and in aliphatic amines206produced

different dechlorinated p h o t o - p r o d ~ c t s . ~However,
~ ~ - ~ ~ ~it requires long irradiation
times (48 to 50 hr) for mirex to disappear under their conditions and as expected many
photolysis products are obtained. In the case of UV photolysis in hydrocarbons (cyclo-
hexane or i ~ o c t a n e ) the
, ~ ~major
~ products are the 8-monohydro- and 2,8-dihydro de-
rivatives, whereas in the presence of aliphatic amines the major products are the 5-
monohydro and two isomeric 5,lO-dihydro derivatives. These workersz05-207 worked
on the macro scale (g) level. At the much lower concentrations normally encountered
in residue analysis, reaction times are greatly reduced, especially when quartz reaction
vessels208rather than borosilicate vesselsm6are used. For example, Lewis et al.'' were
able to effect 95 to 100% photodegradation of mirex at 100 pg/p1 in hexane containing
10% by volume of triethylamine after 15 to 20 min. Although not specifically men-
t i ~ n e d , ~the
O ~ numerous reaction products produced and the similarity in retention
times of some of these products in PCB regions make this direct UV photolytic method
for its confirmation unattractive. Therefore, these authorszo8used an indirect approach
to confirm mirex in the presence of PCBs (Aroclor8-1260). The procedure depends
on diethylamine-assisted photodegradation of interfering PCBs prior to measurement
of the mirex by ECD-GC. Thus, after irradiation for 100 min in a quartz tube, the
heptachlorobiphenyl in Aroclor8-1260 which interferes with mirex is selectively pho-
todegraded while only 0 t o 5 % of the mirex was lost. This procedure was later slightly
modified and investigated further for the confirmation of mirex in Canadian human
milk samples.209These Canadian researcherszo9show that the percentage of mirex re-
covered after UV irradiation varies with PCB (Aroclor8-1260)/mirex ratio. In the
presence of higher concentrations of Aroclor8-1260 the recoveries of mirex were
lower. Their results based on 3 determinations are summarized below:
Vo Mirex
Ratio PCB/mirex recovered
This UV photolytic p r o c e d ~ r e ssimilar ~ ~ ~ . in

~ ~approach
~ as the perchlorination or ni-
tration procedures for the confirmation of PCBs in that the interfering PCBs are re-
moved completely in the chemical procedures or partially in the UV procedures,
whereas mirex virtually remains intact. The presence of mirex peak after chemical and
photochemical reaction is inferred as substantiation to its identity. Therefore, these
procedures can only be regarded as indirect, semiconfirmatory technique because the
presence of mirex peak, although less likely, could be due to an unknown artifact.
Recently, UV irradiation at the surface of mallard duck egg homogenate containing
mirex was reported.210"Irradiation products appeared to be mono and dihydroderi-
v a t i v e ~ " ~as' ~observed by Alley et a1.205-207
in irradiation of mirex in organic solvents.
This is not an analytical or confirmation procedure as one review2I1implied, but rather
of academic interest.
3. Kepone
For the confirmation of kepone there is only one method reported212at the time of
writing this manuscript. It is based on the original perchlorination procedure described
in old patents as quoted by Ungnade and M ~ B e e in l ~their
~ classic review in 1958 which
stated; "The anhydrous ketone Cl,ClloO forms solvates with water, alcohols . . . . On
being heated with phosphorous pentachloride at 125-150°C, it is converted t o a chlo-
rocarbon, Cl,C12 m.p. 485°C identical with the product of self-condensation of hex-
achlorocyclopentadiene with aluminum chloride." The conversion of this kepone to
mirex on treatment with phosphorus pentachloride was also mentioned later by Rob-
In this confirmation procedure212for kepone, mini-column cleanup of the reaction

extract should be an integral part of the confirmation procedure because of the impur-
ities in the reagents and side products generated from the reaction. The procedure has
been applied to shellfish and fin fish extracts.
This perchlorination procedure, to a certain degree, is similar to the perchlorination
of PCBs mentioned earlier. The procedure calls for heating sample extract after solvent
evaporation at 145OC for 3 hr with 200 mg phosphorus pentachloride, 50 mg aluminum
chloride, and 3 m1 carbon tetrachloride in a TeflonB-lined screw-capped tube. The
authors reported a 104% conversion of kepone to mirex with relative SD of 8.2%.
Since the conversion is quantitative and mirex has a larger ECD response than kepone,
this confirmation procedure is theoretically quite sensitive. However, due to the large
amount of by-products and quality of the reagents commercially available, the sensi-
tivity in practice is far from the suggested theoretical sensitivity. Due to the high re-
agent and reaction background, its application to most water samples would be lim-
ited. Although not mentioned in the paper,'I2 this procedure is potentially hazardous,
mainly because of the extreme pressure buildup during the reaction in the reaction
tube and the corrosiveness and reactivity in handling PC1, and AlCl,. All the reaction
tubes used for the reaction must be carefully examined for tiny cracks and imperfec-
tions before use to avoid possible explosion during and after reaction. Protective cloth-
ing and eye goggles must be worn at all times. In addition to the procedure being
hazardous, there is one major practical disadvantage. It is the presence of a consider-
able amount of ECD-responsive impurities and by-products from this reaction. This
is obvious from the broad solvent front of the chromatograms. While this broad cluster
of peaks does not interfere with the confirmation, repeated injections of the reaction
extracts would decrease performance of the GLC system. Therefore, in contrast to the
authors' recommendation that the derivatized extracts could be analyzed without Flor-
isil8 column cleanup, it is not recommended to inject these extracts into the GLC
system under any circumstances without cleanup. Like all reactions using elemental
halogens (such as bromination and chlorination) or using reactive chlorinated reagents
such as antimony pentachloride (as in PCB perchlorination) or aluminum chloride
and phosphorus pentachloride (as in this case), traces of elemental halogen or some
halogenated compounds which are either formed or are intrinsic to the reaction are
difficult to remove completely. It is therefore suggested that using the same GLC sys-
tem for the examination of these reaction extracts as for water analysis should be
avoided if optimum sensitivity of the GLC system is to be maintained. It may be
pointed out that the FlorisilB column chromatography does not effectively remove
those compounds responsible for the large solvent front.
4. Lindane and Other BHC Isomers

The term BHC (benzene hexachloride) is an unfortunate one because it is a chlori-
nated cyclohexane and not a benzene compound. This term is easily confused with
hexachlorobenzene (HCB) which is a different compound. In European countries,
HBC is more accurately and less confusingly referred to as HCH (hexachlorocyclo-
by' ~chlorination of benzene under UV light; hence
hexane). BHC is m a n ~ f a c t u r e d ~
the name BHC was originated. Technical product is predominately a mixture of several
hexachlorocyclohexane isomers containing alpha-, beta-, gamma-, delta-, and epsilon-
isomers with alpha-isomer the most and gamma-isomer the second most predominant
isomers.214 However, the percentage of each isomer varies from manufacturing
Table 23
CHEMICAL CONFIRMATION O F HCB AND BHC
Reaction Known
HCB NaOMe (Na in MeOH) Reflux, 1 % hr Not investigated 228

G C column: 200°C
0.5 m1 (10% KOH I-pro- Boiling water, 10 Some BHC isomers inter- 229
pan01 sd.) + 0.5 m1 pyri- min fere in some columns but
dine G C column: not in others (see text)
190-200°C
NaOMe (Na in MeOH) Reflux, 1-10 hr. Lindane does not interfere 239
G C column: 135°C
0.2 N K O H in ethylene gly- Heat 150°C, seal No interference from 0.c.s 225
col, heated and methylate tube, 1 hr. methy- and PCBs
late with diazome-
thane,
G C column: 150°C
0.2 m1 pyridine, 0.5 m1 Boiling water, Not completely known (see 230
10% KOH in 2-propanol 30-60 rnin, text)
and 3 other R O H G C column: 200°C
0.2 m l pyridine, 0.5 m1 Application of Crist Application of Crist et 23 1
10% KOH in 2-propanol et al.'30 aI.l3O method
a n d 3 other ROH G C column: 180°C
BHC isomers 2 % KOH in ethanol 100°C. 15 min. BHC isomers mutually in- 85,86
(a, P, u. 4 G C column: 200°C terfere
BHC isomers 0.1 g NaOMe in MeOH (2 60°C, 15 min. BHC isomers mutually in- 216
(a.P,u) ml) G C column: 200°C terfere
sources. For example, Stijve and Cardinalez's found that a Japanese BHC contains
10% of the beta isomer (instead of 3 to 5%30s),whereas a sample of American origin
revealed a gamma-BHC content of not less than 40% (instead of 13 to 16%). In any
event, alpha-BHC is still the most predominant isomer in the technical product. Of
the eight theoretically possible stereoisomers, only the above five are significant in
technical BHC. Among these isomers, the active ingredient is the gamma-isomer (lin-
dane),2i4used as an insecticide. (See Chapter 2, Section I for structures of the impor-
tant BHC isomers.)
A t present Cochrane and MayburyZi6are the only authors to report a concrete chem-
ical derivatization procedure specifically developed for lindane and two BHC isomers
(alpha and beta) although several papers mention or discuss dehydrochlorination of
BHC (see also Table 23). For example, Stijve and Cardinale in 1972 reported the use
of neutral A1203TLC plates6' to degrade a number of BHC isomers at 60°C for 45
min at milligram level to trichlorobenzene isomers. The yield of the AlzO3 dehydro-
chlorination procedure was not better than 20% for each isomer. They also used a 0.2
N ethanolic KOH for micro-scale preparation. Krausess used 2 m1 of 2% ethanolic
KOH at 100°C for 15 min for the quantitative determination of perthane while also
screening 40 o r so other pesticides including 4 BHC isomers (alpha, beta, gamma,
delta) and in each case observed complete degradation to afford the same product
having a very short retention time very close to the solvent front (GC column: 200°C).
Based on the retention time datas5 and the well-known chemistry of BHC dehydro-
chlorination (see discussion later), this peak is probably the 1,2,4-trichlorobenzene.
With the minor and insignificant modification of using 1 m! instead of 2 m l ethan-
olic KOH, Young and Burkes6 used a dechlorination procedure identical to that of
Krauses5 to investigate many pesticides. Theyzi6also observed that "the alkali treat-
ment completely eliminated lindane, the alpha, beta, and delta isomers of BHC."
However, "only small early eluting gas chromatographic peaks, presumably from tri-
chlorbenzenes, were observed." These low recoveries of trichlorobenzenes are dis-
cussed by Cochrane and Maybury216(see discussion later).
The method of Cochrane and Maybury216involves treatment with sodium methoxide
in methanol (NaOMe/MeOH) at 60°C for 15 min for the confirmation of lindane and
the alpha and beta BHC isomers in cereal, animal feeds, meat, and fat samples. This
procedure can detect 0.01 ppm lindane in a 10-g sample. The authors do not specify
the detection levels for the other two isomers.
Reaction in solution is also comparedz16 to the alkaline precolumn technique of
Miller and Wells40 discussed earlier in this chapter. Although the alkaline pre-column
technique saves time, Cochrane and Maybury216found that the pre-column technique
does not eliminate interferences in some meat and fat samples as in the reaction with
NaOMe/MeOH.
Alkaline dehydrochlorination of BHC is very well documented since 1833. Davidow
and WoodardZ17pointed out that "Mitscherlich loc. cit. (Ann. de chim. et de phys.,
55, 41, 1833) in 1833 described the formation of trichlorobenzenes from benzene hex-
achloride by treatment with alkali. Van der Linden218in 1912 studied the products of
alkaline hydrolysis of alpha, beta, and gamma isomers of benzene hexachloride and
by a method of mixed melting points estimated that of the mixture of 1,2,3-, 1,3,5-,
and 1,2,4-trichlorobenzene formed, approximately 82 per cent was the 1,2,4-trichlo-
robenzene." Bradbury and Standen2l9 further commented on the work of Van der
Lindenz18 that the proporions of the three trichlorobenzene isomers differ slightly ac-
cording to the BHC isomer and the dehydrohalogenating agent used, but in all cases
1,2,4-trichlorobenzene was the major product. Thus in the case of the NaOMe/MeOH
procedure,216all the 3 BHC isomers studied produced 1,2,4-trichlorobenzene as the
major product with 1,3,5- and 1,2,3-trichlorobenzene as secondary or minor products.
The main difficulties of using the dehydrochlorination approach, disregarding the al-
kaline systems used, is the volatility of the products (trichlorobenzene). Cochrane and
Maybury216 found that up to 100% was lost due to volatilization of these relatively
low melting trichlorobenzene isomers during evaporation of the residual solvent and
suggest (but giving no details) the use of fractionation distillation to remove the meth-
anol in the reaction mixture before extraction with benzene. If methanol is present,
even after dilution with water, variable losses of trichlorobenzenes were ~ b s e r v e d . " ~
The amount of loss depends on their concentration. In addition to this variation, con-
firmation of BHC by any dehydrochlorination procedures is inconveniently carried
out because it requires a lower GC column temperature (150 to 180°C) to analyze the
trichlorobenzenes than the usual column temperature (200 to 210°C) for 0.c.s and
PCBs analysis. In other words, a separate GC has to be set aside for the confirmation
of BHC by the dehydrochlorination procedure. As we all know, the GC system is a
temperamental instrument. It is inadvisable to switch frequently from one set of oper-
ating parameters to another; for some reason, the original performance may not be
maintained. Therefore, in a routine laboratory, there is a tendency to leave the GC
system alone if it operates efficiently. As discussed in Chapter 2 for O.C.analysis, the
region in a chromatogram from the solvent peak to lindane or aldrin peak is quite
often plagued with interfering sample CO-extractivepeaks. The first portion of this
region is where the trichlorobenzene isomers appeared, due to their shorter retention
time than lindane. Fortunately, Cochrane and Maybury216make a good choice of de-
hydrochlorinating reagent (NaOMe/MeOH). This stronger alkali is more effective in
reducing or even eliminating some of the sample CO-extractivethan the KOH/EtOH
system used by KrauseE5or Young and B ~ r k e . "However,
~ it should be noted that this
effect of the stronger alkali reagent (NaOMe) could be decreased by the milder dehy-
drochlorination conditions (60°C, 15 min) as compared to the more drastic conditions
(lOO°C, 15 min) Krause or Young and Burke used. Depending on sample types and
sample cleanup efficiency, CO-extractivescan still occur in this region.
As may be already obvious the dehydrochlorination p r ~ c e d u r e s "require

~ the BHC
isomers to be pre-separated before reaction; otherwise, it is difficult to interpret the
results, since as mentioned above, all three BHC isomers after alkali dehydrochlori-
nation in solution give 1,2,4-trichlorobenzene as the major product and 1,3,5- and
1,2,3-trichlorobenzenes as secondary products, or the same unidentified product from
alkaline pre-column procedure (Figure 1 of Reference 216). These three BHC isomers
(alpha, beta, and gamma) are eluted in the same fraction (6% ether in hexane) from
the MillsI5' FlorisilQ column cleanup procedure but we found that alpha-BHC and
lindane can be easily separated using Reynolds' FlorisilB column cleanup procedure
(Table 6). However, Crist et al.230observed the various isomers of BHC were not eluted
by hexane (see discussion later). Unfortunately we did not investigate the other BHC
isomers, particularly the more significant beta-isomer. It is expected that these other
BHC isomers (beta, delta, and epsilon) will be CO-elutedwith either the alpha-isomer
or lindane; hence it may not be easy to separate them into single fraction from each
other. This is the major difficulty in applying any one of the dechlorination procedures
discussed for the confirmation of BHC isomers in actual samples. The question is how
to apply any one of these procedures to actual sample extracts. In a real life situation,
samples can contain many 0.c.s; in particular, most or all the BHC isomers can be
present. Although the paper of Cochrane and Maybury216 is the most specific and
detailed work so far published for the chemical confirmation of BHC isomers in real
samples, surprisingly there is no comment on this major difficulty. It is not readily
apparent how these authors analyzed and confirmed the three BHC isomers they in-
vestigated in cereal, animal feed, tissue, and fat sample if they all occur in the sample
extract. Further, if the other BHC isomers are also present, giving the same dehydroch-
lorination product, how would one distinguish one BHC isomer from the other? Under
the extraction section, these authors stated "cereal and animal feed samples were ex-
tracted and cleanup [was] according to the ball-mill procedure of Levi et al."22o and
"the meat and fat sample by low temperature cleanup technique of McLeod and
Wales."221 The latter method does not discuss the separation of BHC isomers whereas
Levi et al. eluted all the 0.c.s investigated including BHC and lindane into one fraction
from a 2% deactivated FlorisilO column rather than separating the 0.c.s into frac-
tions. PCBs would also be in this fraction. These authors did not mention what BHC
isomers they investigated but use the term BHC separately from lindane - presumably
it is the technical BHC. Therefore; these two cleanup procedures quoted by Cochrane
and MayburyZfi6 are not compatible to the NaOMe/MeOH or the alkaline pre-column
procedures for the confirmation of lindane and other BHC isomers if all are present.
in the same sample extract.
Beta-BHC is known to be very resistant to dehydro~hlorination.~'~ Specifically,
Cochrane and Maybury216observed that "in the dehydrochlorination of lindane, quan-
titative conversion to trichlorobenzenes (in NaOMe/MeOH) was achieved within 10
min at 60°C. Similiarily, a-BHC was derivatized within 5 min, while p-BHC was not
affected after 15 min reaction period." However, the chromatogram on Figure l2I6
and the tone of the paper indicate that all three BHC isomers are dehydrochlorinated
by their procedure. Therefore, it is not apparent whether p-BHC is reacted or not as
described by the procedure (60°C for 15 min). If 0-BHC were indeed not affected,
then the described procedure will only derivatize a-BHC and lindane. Under this situ-
ation, confirmation of these two BHC isomers would be easier in those samples con-
taining BHC isomers because, as mentioned previously, lindane and a-BHC can be
easily separated by a FlorisilB column (Table 6) before dehydrochlorination. Thus
they no longer interfere with each other and the unreacted p-BHC has a longer reten-
tion time than the trichlorobenzenes. Interferences from the other isomers (delta and
epsilon) are still potentially there. However, since they are only minor products in
technical BHCZ14.215 and unlike lindane ()I-BHC), are not used individually as insecti-
cides, their contribution to the interference of other BHC confirmation is expected to
be less significant.
Instead of alkalinic dehydrochlorination, Dennis and Cooper92.222 investigated de-
chlorination of lindane by the reaction of nickel boride (Ni2B), prepared in situ by the
reaction of sodium borohydride with alcoholic nickel chloride. They found that "com-
plete conversion of lindane to a mixture of benzene, cyclohexene and chloride ion
could be achieved in 15 minutes."222 Benzene was the major product. However, this
approach has not been evaluated on sample or sample extract. Due to the volatility of
the products this procedure would be less convenient in routine application. Another
confirmation procedure, although beyond the scope of this manuscript, is worth men-
tioning. It is a physical method using high speed liquid c h r o m a t ~ g r a p h yspecifically
~~~
addressed to the problem on the confirmation of lindane in routine application. This
method has diagnotic value for lindane because its elution pattern is reversed from
FlorisilB column chromatography or many commonly used GLC columns.
Due to the reversed order for the elution of lindane on a 3% OV-225 column, it has
been this author's favorite since 1970 for providing additional substantiation for the
identity of lindane. Thus, instead of the usual elution pattern (lindane, aldrin, hepta-
chlor) in commonly used mixed phase GC columns such as 4% OV-101/6% OV-210,"'
" 11% " OV-17/QF-l (see Chapter 2) and 4%SE-30/6% QF-1, lindane elutes after hep-
tachlor in a 3% OV-225 column. However, as mentioned earlier in this chapter and

by several authors (see Section I) multi-column technique alone cannot be considered
as confirmation of identity.
5. HCB (Hexachlorobenzene)
Unlike BHC (or HCH), HCB (1,2,3,4,5,6-hexachlorobenzene) is a chlorinated aro-
matic compound and hence is more resistant to dechlorination. (Elementary organic
chemistry tells us that aromatic chlorine is much more inert than aliphatic chlorine.)
In fact, StijveZL4stated that HCB "is not destroyed by treatment with sulphuric acid
and studies performed in our laboratory indicated a high stability towards alkaline
substances. In fact, we think that its stability may be compared with that of the noto-
rious beta-HCH which is the most persistent of all hexachlorocyclohexane isomers."
HCB is prepared by catalytic perchlorination of It is used as a fungicide
but as pointed out by H ~ l d r i n e itt ~is~also
~ a by-product from such organic synthesis
as the production of pentachloroethylene and vinyl chloride.226As pointed out by other
HCB is also used as a starting material for the production of pentachloro-
phenol (a wood preservative). For a brief but informative discussion on the history
and background of HCB, see Stijve's paperz15of 1971.
Similar to BHC (HCH), alkalinic dehydrochlorination is a popular chemical deri-
vatization procedure for HCB (Table 23). Taylor and Keenan228in 1970 investigated
either alcoholic potash or sodium methylate for the derivatization of HCB and found
the latter reagent more dependably effective. It is understandable that, being a stronger
alkali, sodium methylate will react with HCB faster than KOH under similar condi-
tions. Thus, these authors found that at 0.1 ppm or less of HCB the pattern of degra-
dation is consistent t o give two products (one with the same retention time as HCB
and the other with shorter retention time). However, "when larger amounts of HCB
are treated the result is often obscured by the appearance of several other products."228
These authors therefore concluded that "the reaction is not a reliable confirmatory
test for all samples." Disregarding that their procedure has this major shortcoming,
the straightforwardness and apparently subjective style of this paper is admirable.
After reading it, the reader knows what to expect from their method. This paper does
not attempt to hide the weakness of the proposed procedure, exaggerate the claim,
and even report results of others out of context to improve the results of the authors'
own investigation. It must be realized that pointing out the weakness as well as the
strength of one's own procedure not only assists the reader tremendously rather than
confusing him, but also reflects the thoroughness of the author's investigation and
discussion.
As shall be discussed later, the extraction procedure for the reaction product by
Taylor and KeenanZz8reflects the practical experience and attention to small but im-
portant details necessary for smooth routine G C operation.
Collins et al.229used freshly prepared KOH in l-propanol in the presence of 0.5 m1
pyridine to react HCB for 10 min at boiling water temperature. An ether, identified
as pentachlorophenyl propyl ether (monopropoxylpentachlorobenzene), was obtained.
This ether can be analyzed at a GC column temperature of 190 to 200°C (a convenient
feature as discussed previously) and has longer retention time than the parent com-
pound (HCB) and three BHC isomers (a, y , and 0) in the two mixed GC columns
investigated by these authors. Unfortunately, the ECD response of this ether derivative
as compared to HCB or the detection level of the procedure was not presented. How-
ever, Crist et al.230later extended this procedure and investigated in more detail to
include three more alcohols (ethanol, 2-propanol, and butanol) in addition to the 1-
propanol.
"The two most useful compounds were isopropoxypentachlorobenzene (IPB) and
bis-isopropoxytetrachlorobenzene(BITB) derived from 2-propanol. The latter product
appeared to be less subject t o further substitution than the former and the reproduci-
bility of the reaction was more easily controlled."230 Therefore, although the forma-
tion of the monosubstituted ether (IPB) derivative requires much shorter reaction time
(10 min) than the disubstituted (BITB) derivative (30 to 60 min), Crist et al.230favor
the latter derivative for the confirmation of HCB. The use of IPB and other ether
derivatives from reacting HCB with other alcohol offers additional options when at-
tempting to confirm HCB in the presence of several interfering peaks. These authors
described one example that in the presence of Arochlor@ 1016 (CO-elutedwith HCB
in the hexane fraction from Florisil@ column) "derivatization of HCB to BITB was
not feasible due t o interference from a later eluting Arochlor@ peak." However, they
stated that "it was possible to confirm and quantitate HCB (0.3 ppm) based on the
monosubstituted derivative (IPB) which (having a shorter rentention time) was well
separated from other components in the samples." Although not mentioned in the
text,z30besides having an ECD response of approximately 70% that of HCB, BITB
has a longer retention time. Therefore, it can be readily analyzed by the usual GC
conditions (e.g., oven temperature 200°C) for O.C.and PCBs analysis without chang-
ing GC parameters. Unfortunately, Crist et al. do not mention detector responses of
IPB and other ether derivatives. Crist et al.230integrate the standard Florisil69 cleanup
and fractionation procedure in their confirmation procedure to remove possible inter-
ference from CO-extractivesor other pesticides. However, these authors observed that
"the various isomers of hexachlorocyclohexane (BHC) were retained by the FlorisilO
after hexane elution. We found that hexane elutes a-BHC (Table 6) and
HCB233using factory calcined FlorisilG9 kept at 130°C for overnight before use. This
observation has been repeated several times with different batches of Florisil@. Also,
heptachlor, aldrin, p,p'-DDE (and not o,p'-DDT) were found233to be eluted com-
pletely by hexane as expected from Reynolds' procedure.88 The explanation for this
discrepancy could be the difference in amount of FlorisilO, volume of hexane, and
activity of FlorisilB used by these authors and by us.
As discussed previously in this manuscript, a FlorisilB column such as that used in
the Reynoldses procedure is one of the commonly used cleanup and fractionation tech-
niques in 0.c.s analysis. Heptachlor and a-BHC are CO-eluted with HCB in hexane in
the Reynolds procedure. It is desirable to know whether heptachlor, a-BHC, or its
products from the derivatization procedure could interfere with either the IPB or BITB
derivatives. This aspect is not discussed in this paper although it was mentionedZ3Othat
"none of the unchanged pesticides interfered gas chromatographically with either IPB
or BITB." The practical applicability of this confirmation procedurez30 was further
demonstrated by B r e ~ i k in ~ ~1978
' using 2-propanol (isopropanol) and potassium hy-
droxide.
Baker'sZ3l investigation of the confirmation of HCB by chemical reaction involves
its reaction with sodium ethoxide in hexane at reflexing temperature. The chemistry
of this reaction is similar t o the reactions studied by Collins et al.229and Crist et al.230
An ether derivative (monoethoxypentachlorobenzene) is formed. Unlike the observa-
tion of Crist et al.230that prolonged reaction gives disubstituted ether as the major
product, Baker232did not mention the formation of any disubstituted ether; even the
reaction was carried out for several hours (1,2,4,6 and 10 hr). Only the monosubsti-
tuted ether was observed as the predominant product. The procedure of Bakerz3' re-
quires at least 8 hr to react most of HCB to the ether derivative. The lack of pyridine
as a catalyst as used p r e v i o ~ s l is
y ~one
~ ~possible
~ ~ ~ ~explanation for the slow reaction.
Another explanation could also be the low boiling point of hexane used in the reaction
medium23zto prevent a higher reaction temperature being attained as in the case of
Crist et al.z30or Collins et al.z29Finally, hexane does not mix with ethanolic sodium
ethoxide solution; this HCB (in the hexane layer) is not constantly exposed to the alkali
reagent. This causes very slow reaction and ineffective derivatization as the author
observed.z3zExcept for lindane, the author did not comment upon or investigate in
this study whether other pesticides or their degradation products, if also present, could
interfere with the confirmation procedure. In view of the above discussion, this pro-
cedure is not suitable for routine application.
H ~ l d r i n e confirms
t~~~ the HCB by reaction with KOH in ethylene glycol at 150°C
for 1 hr in a well-stoppered tube sealed by masking tape. (The use of masking tape
may look crude, but it works. We also used it in some CrClZconfirmation procedures.)
After this reaction, the reaction extract is methylated by diazomethane before GLC
examination. As pointed out by the author,225HCB is first converted to pentachloro-
phenol which is then methylated to the methyl ether. This method has two disadvan-
tages: (1) because two derivatization steps are involved, it is more lengthy than the
procedure of Crist et al.,230and (2) the short retention time of the methylated derivative
necessitates a lower GC column temperature than that commonly used for 0.c.s and
PCBs analysis. Thus, a separate GC is needed for this procedure. However, there are
also several advantages in her procedure. In fact, it is one of the better procedures for
chemical confirmation of pesticide residue simply because it contains sufficient infor-
mation and data to enable the reader to apply this procedure to the actual sample
without wondering, for example, whether the procedure works for the lower level
founded in samples, o r whether the presence of other 0.c.s or PCBs could interfere.
Since the cleanup procedure is discussed in detail, a complete package is presented to
the reader for immediate application. The most important aspect is that the described
cleanup and fractionation procedure is compatible with this proposed confirmatory
method. For example, the possible interferences from common 0.c.s and from PCBs
usually encountered in samples are eliminated by the described cleanup procedure
which isolates HCB for analysis and subsequent chemical confirmation. The author
also tests her chemical confirmation procedure at realistic levels spiked in actual sam-
ple. Similar to Taylor and Keeman,2z8H ~ l d r i n e was t ~ ~also
~ more attentive to details
in the extraction procedure for the reaction products than many other authors such as
Crist et Particularly for an alkaline reaction, improper extraction procedure is
deleterious t o the GLC system. For example, if the reaction extract is not washed well
with water, or dried thoroughly with sodium sulfate, repeat injections of these extracts
on a routine basis with cause poor GC performance due to the presence of traces of
water and alkaline substance in the extract causing slow deterioration of GC perform-
ance. Therefore, for most chemical confirmation reactions and all those involving al-
kali, it is necessary to wash the organic reaction extract well with water to remove
traces of alkali (sometimes addition of acid to decrease alkalinity is advantageous) and
the washed extract dried over anhydrous sodium sulfate before injection into GC. This
small but important detail appears to be easily overlooked by many researchers as
evidenced by close examination of their extraction procedures. Admittedly, procedures
of some of the early papers of this author have this fault. In the extreme case, some
researchers describe extraction procedures in which the reaction mixture is merely
shaken with organic solvent and injected into the G C without washing with water and
drying over Na2S04. Depending on the reagent and reaction type used, washing the
reaction extract with water before drying over Na2S04could be omitted (such as in
the case of CrC12 reduction) but invariably when alkali reagent is used, both treatments
are necessary to prevent deposition and accumulation of minute traces of alkalinic
substance onto the G C inlet or column upon repeat injections. This substance, as men-
tioned in the section for DDT dehydrochlorination, will eventually deteriorate GC per-
formance. This is particularly critical in ultra-trace analysis as for natural water sam-
ples. However, in a research laboratory, this phenomenon may not be realized since
the G C system is not as intensively used for injections of similiar extracts as in an
operational laboratory.
In summary, even though Holdrinet's procedure is somewhat longer, it is a very
thorough investigation for the analysis and confirmation of HCB, readily adaptable
to routine application. The more convenient procedure of Crist et al.230is a good alter-
native, but it could be subjected to interference from PCBs and other 0.c.s unless
asuitable cleanup and residue fractionation procedure is integrated with this confir-
matory procedure. This aspect is only partially addressed by these authors. There is
one small but important point that the reader should be aware of in using the proce-
dures of Crist et al.230and Collin et al.224In particular, there is no provision to remove
traces of pyridine in the organic (hexane) extract before GLC examination. As pointed
out elsewhere,234"the use of pyridine is undesirable for the following reasons: pyridine
is more difficult t o purify requiring several distillations, depending on the source and
impurity present, before it can be used for low level determination (of trace organics);
pyridine must be removed from the organic extract after reaction otherwise broad sol-
vent fronts result, as observed on occasions when concentration of the extract was
required before GLC determination." Even if the reaction extract is not to be concen-
trated before GLC determination, traces of pyridine will eventually affect GC perform-
ance. T o remove pyridine, aqueous HCl is added to form a water-soluble pyridinium
salt and traces of HCl in the organic extract is removed by washings with water. Fi-
nally, drying over anhydrous sodium sulfate removes water and hence any possible
traces of acid that is not removed by the washing. (See also the detailed procedure in
treating organic extract from an alkaline reaction as described by Taylor and Kee-
man.228)
For those who use a FlorisilB column for sample cleanup and pesticide fractionation
rather than the charcoal column described by H ~ l d r i n e t the
, ~ ~hexane
~ fraction could
be used for chemical derivatization, since HCB is eluted in this fraction. Possible in-
terference from 0.c.s and PCBs also eluted in the hexane fraction (Table 6 ) can be
eliminated if some minor modification is incorporated into the H ~ l d r i n e procedure.
t~~~
Thus after reaction of HCB with KOH in diethyl glycol, and dilution of the reaction
mixture with 10 m1 saturated Na2S0, as described,12' the aqueous solution is extracted
twice with hexane or benzene t o remove the neutral pesticides (PCBs, o.c.s, and any
reaction products formed). The HCB derivative (pentachlorophenol) remains in the
Volume I 131
alkali aqueous solution, which can then be acidified by H2S0, and extracted with ben-
zene as described by H ~ l d r i n e tAlternatively,
,~~~ one could consider incorporating the
method of Chau and C ~ b u r for ~ ~analysis of pentachlorophenol. This procedure
n ~the
does not require the extraction of pentachlorophenol into organic solvent before deri-
vatization, since unlike methylation with diazomethane, acetylation can be carried out
in the aqueous alkalinic solution. However, it has not been investigated whether the
presence of ethylene glycol or saturated sodium sulfate solution used in Holdrinet's
procedure has any effect on the aqueous a ~ e t y l a t i o n . ~ ~ ~
IV. OTHER PESTICIDES
A. Organophosphorus Pesticides (0.P.s)

The number of chemical derivatization procedures available for the confirmation of
0.p.s is much less than that for 0.c.s. As shown in Tables 25A and B and Table 26,
the procedures published so for for this purpose can be classified into 2 main ap-
proaches: (1) hydrolysis of 0.p.s and derivatization of hydrolytic products containing
P moiety (Tables 24 and 25A) or containing phenolic or thiophenolic moiety (Table
25B), and (2) derivatization of intact insecticides (Table 26).
l . Hydrolysis o f 0 . P . s and Derivatization o f the Products

a . Derivatization o f the P Moiety
This approach is based on the facile hydrolysis of most 0.p.s by alkali (see discussion
in Volume 11, Chapter 2). Table 24 summarizes the most frequently encountered P-
containing hydrolytic products. Many 0.p.s have the following general structure:
RO\ S(or 0 )
/ p\
R'O S(or 0 ) X
Usually the alkyl groups (R) are the same (R=R') and are either methyl or ethyl. The
two RO-groups (dimethoxy or diethoxy) and the 0=or S= bonds are stably linked to
the P atom but the -SX or -OX bond is easily cleaved by alkaline hydrolysis:
R0 S(or 0 ) R0 So10
\p/ OH@
'
A \ p + HSX (or HOX)
RO/ \ or O)X RO/ OH
Since most 0.p.s are esters of phosphoric, phosphorothionic, phosphorothiolic, and

phosphorothiothionic acids, only two types of P-containing products (Structure 1)
namely, dialkyl phosphate and 0,Odialkyl phosphorothionate are generally encoun-
tered as a result of hydrolysis. Since dialkoxy groups (RO-) are generally either dime-
thoxy or diethoxy group, four P-containing hydrolytic products are usually formed.
These are dimethyl phosphate, diethyl phosphate, dimethyl phosphorothionate, and
diethyl phosphorothionate (see Table 24).
Several authors developed screening or confirmation procedures based on the alka-
line hydrolysis to the corresponding dialkyl phosphates or 0,Odiphosphorothionates
which are methylated by diazomethane to the corresponding volatile trialkyl deriva-
tives suitable for GLC analysis. St. John and Lisk23sused this approach for the quan-
titative determination of 5 0.p.s and Askew et al.236expanded this approach to 32 0.p.s
and metabolites. Shafik et optimized the hydrolytic conditions of 32 0.p.s result-
ing in three main methods based on the use of different amounts/concentrations of
NaOH for hydrolysis. For better separations of derivatives both methylation and ethy-
lation (by diazoalkane) are used.
Table 24
COMMONLY ENCOUNTERED P-CONTAINING HYDROLYSIS PRODUCTS
R0 0 R0 0 R0 0
\p/ \p/ \ /
or
/ / \ /p\
R0 \O-X R0 S-X R0 OH
Phosphate Phosphorothiolate Dialkyl phosphate
R0 S R0 S R0 S
\p/ or \p/
RO/ \ 0-X RO' \ S-X RO' 'OH
Phosphorothionate Phosphorothiolothionate Diakyl

(phosphorothioate) (phosphorodithioate) phosphorothionate
As pointed out by Shafik et those 0.p.s hydrolyzing to dimethyl phosphate

could not be confirmed in some sample matrices by the procedure of Askew et al.236
because of interference from naturally presented inorganic phosphate which also yields
trimethyl phosphate under the conditions of analysis. "Another major limitation to
the use of alkaline hydrolysis as a route to confirmation of the identity of 0.p.s has
been the inability to find a GLC column which satisfactorily resolves all of the trialkyl
phosphates of interest."237 This problem is said237to be resolved by "systematically
varying hydrolysis conditions to select either the most complete hydrolysis or the most
characteristic products and by using a 20% Versamid column for separating mixtures
of alkyl phosphates." However, it is not always practical to apply their methods if
several 0.p.s CO-existin the sample extract unless the extract is split into several por-
tions to be analyzed by the various mentioned hydrolysis and alkylation procedures.
If so, the sensitivity of detection will decrease and analysis time will increase.
Due to the nature and volatility of the methylated derivatives, the GLC operating
conditions for their examination are necessarily quite different from those normally
used for the analysis of parent pesticides. For example, the column type is different
and is only suitable for examining the derivatives rather than the parent 0.p. com-
pounds or most other pesticides; the column temperature is much lower than the usual
200°C for pesticide analysis. Consequently, a separate GLC must be dedicated to this
screening procedure. This inconvenience in GLC operation is another major practical
disadvantage in addition to its nonspecificity.
For a recent discussion on the use of methylation reaction for gas chromatographic
determination of 0.p.s the reader is referred to Reference 238.
Among the various methylating reagents (BF3/MeOH, HCl/MeOH, methyl fluoro-
sulfate, CH31/K2C03,dimethyl sulfate), diazomethane is most widely used for methy-
lation of the P-containing alkalinic hydrolytic products. In fact, it was commented211
in a study that "methylation of phosphonic acids such as e t h e p h ~ n 'or ~ ~the desmethyl
derivatives of Oaryl-0,Odialkylphosphorothioates is best achieved using diazome-
thane" as the other methylating agents239were found ineffective or tedious.
Rather than the two-step procedure, hydrolysis and methylation can be conveniently
performed in one step by "inlet block" m e t h ~ l a t i o n . ~ ~Th O us
- ~ ~the
~ 0.p. pesti-
CideS241.242are simultaneously hydrolyzed and methylated by alcoholic trimethylanili-
nium hydroxide (sometimes referred to as trimethylphenylammonium hydroxide
[TMAH]) at 280°C in the GLC inlet block. In short, it is a reaction GC procedure.
These methods are probably inspired by previous work on inlet block GLC derivati-
zation such as those of Moye for on-column transesterification of Nmethyl carba-
Volume I 133
Table 25A
CONFIRMATION OF 0.P.s AFTER HYDROLYSIS AND DERIVATIZATION
OF P MOIETY
Reaction type Compounds Reagents Products Ref.
235,236,237,238
S
Hydrolysis and OH- S
methylation R0 CHA, RO\ II
P'- OX / P-OMe
/
R'O RIO
S
RO\ II
/P-SX
R'O
R0 ::
\ P-OMe
/
R'O
""\ 7
P-SX
R'O /
RxGLC (inlet as above TMAH 280°C, GLC inlet block 240-242,243-246

block methy-
lation)
Table 25B
CONFIRMATION OF 0.P.s AFTER HYDROLYSIS AND
DERIVATIZATION OF PHENOLIC MOIETY
Reaction type Compounds Reagents Products Ref.
Hydrolysis and S F
, 254
ether formation F"o\ ; - o + ,
/
~ ' b
TroleneO, parathion, methyl OH-

parathion, cyanox, fenitro- PFBBr
thion, dichlorofenthion
Crufomate (RueleneO)
Dyfonate
Table 26
CONFIRMATION OF 0 . P . s BY DERIVATIZATION OF INTACT COMPOUNDS
Reaction type Compounds Reagent Products Detector Ref.
. Oxidation RIO S R'O S [NaClO] in acetone

room temp. 5 min
R'O
\
0
R'0\
0
II
FPD 25 8
\ p-OR"' \ ;-SRrtt P-SR"'
/
~"0' R"0 R"O/ R"O/
Diazinon, RonnelO, parathion, methyl

parathion, fenitrothion, malathion
Reduction
RIO S
\ 11
aq. CrCl, in ben- P-0-C, H,-p-NH,
zene; 60°C. 10 rnin R"O/ FPD, AFID, ECD 259
Parathion, fenitrothion, E P N methyl para-

thion
Fenitrothion, methyl parathion, parathion aq. CrCI, in ben- CCD
(and NO2-containing herbicides and fun- zene, room temp. 1
gicides) min
Alkylation R,R,P(O)NHR,, R,R,P(S)NHR, (phos- NAH/CH,I/DMSO 0 S AFID

(methylation) phoramidates, phosphoramidothioates) 50°C, 10 min
n II
R, R,-P-NR, , R , R,-P-NR,
Cruformate, Bay 9382C and oxon, dime- I I
thoate CH, CH3
Trifluoro-acety- TFA (0.2 ml), 0.6 0 FPD

0 CH, H 0 H
lation II I I II, m! benzene under II
(CH, O), P--0- C = C- C
N1
\ / C-CF3
CH3 R -N
\
CH,
DasanitO and oxon, nemacur sulfoxide, TFA, 30°C 15 min, AFID
photate sulfoxide, oxydemeton methyl, sealed tube See text
Counter sulfoxide
Volume I 135
3
D
E
<
nf
u
u
a* ^
nU w
f1
fcc: *
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r-
> ^
= -'
•s ^
5 u"
C cfl T3
'J j:
J 8 S>>
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mate243and of o r g a n ~ p h o s p h a t e particularly
,~~~ the work of Tanaka and Wien,14' who
were first to apply trimethylanilinium hydroxide reaction for the derivatization of sub-
stituted phenylureas by flash-heater methylation technique. It may be pointed out
Miles and Dale overlooked the earlier work of Tanaka and Wien in 1973 and mistak-
enly stated that "the first use of TMAH for analysis of a pesticide was reported by
Dale et al. (1976)."240 Interesting to note, in 1976, Bromilow and Lord246also reported
the use of TMAH for the formation of derivatives by on-column methylation (reaction
GLC) of S-containing carbamates.
The terms "flash-heater derivatization," or "on-column," "inlet block", and "in-
block" G C derivatization are interchangeably used by various authors. Perhaps the
term "inlet block" and "in-block" GC derivatization is less informative and more
confusing than "flash-heater" or "on-column'' derivatization t o describe a reaction
GLC procedure whereby the pesticide is pre-mixed with reagents before injection and
reaction in the GLC, as in the case of TMAH derivatization.
The term "inlet block" or "in-block" could easily be confused with the so-called
GLC pre-column technique in which nonvolatile reagents are deposited on a short
column t o react with the injected pesticide (e.g.,, DDT dehydrochlorination by an
alkaline pre-column as described previously). However, there is no agreed upon ter-
minology to differentiate two types of reaction GLC.
Methylation procedure by reaction GLC is more convenient than those carried out
in solution. However, the work of Churchill, Miles, and Dale,24'-242 except the paper
on c h l o r o p h o ~ i mas~ far
~ ~ as practical applicability is concerned, can only be regarded
as preliminary investigation since more procedural refinement and details are needed
before it can be applied to real life situations, particularly for low levels of 0.p.s in
environmental samples. The influence of sample CO-extractivesand the presence of
several 0.p.s on the efficiency and reproducibility of this "in-block" derivatization
technique have yet t o be explored. Nevertheless, it is a potentially good approach to
keep in mind for some specific future practical application.
Disregarding whether solution derivatization or reaction gas chromatographic tech-
nique is used, the approach to derivatize the P-containing hydrolysis product is non-
specific for 0.p. confirmation since as already mentioned several 0.p.s can give the
same derivatives. Therefore, strictly speaking, this approach can only be regarded as
screening rather than confirmatory procedures.
b. Derivatization o f Phenolic or Thiophenolic Moiety

As mentioned above, an 0.p. is hydrolyzed into two products: the P-containing
product and the non-P-containing product. (Depending on the structure of the o.p.,
sometimes a third product, formaldehyde, is formed as in the case of hydrolyzing
phorate or carbophenothion - see Volume 11, Chapter 2, Section IV.) If the non-P-
containing hydrolytic product is a phenol or thiophenol, it can be used for derivative
formation. Since many commonly used 0.p.s have these moieties and most of them
are different, this approach is much more specific than the one using the P-containing
moiety for derivatization. The other advantage of this approach is the number of dif-
ferent reactions available for derivatization of these phenols or thiophenols.
Among the various derivatives used for this purpose, the procedure forming penta-
fluorobenzyl (PFB) ether derivative^^^'-^^^ 1s the most ~ e n s i t i v e compared
' ~ ~ ~ - ~ ~with
~
some other derivatives because these derivatives have large ECD response. The lowest
level of detection of these derivatives can be equal to and even exceed the detection
levels of the corresponding parent 0.p.s as determined by either flame photometric
detection or an alkali flame ionization detector (AFID). Recently the confirmation of
Ronnel, rufomate, methyl parathion, fenitrothion, parathion, DyfonateB, dichloro-
fenthion, and Cyanox were achieved by this a p p r o a ~ h Briefly,
. ~ ~ ~ the
~ ~parent
~ ~ 0.p.s
are hydrolyzed by an alkaline solution, and the phenols or thiophenols are derivatized
to form the corresponding PFB derivatives. The PFB derivatives are fractionated on
a silica gel column to simplify subsequent chromatographic examination and to remove
excess reagents. Using this technique concentrations as low as 0.01 to 0.1 ppb of these
pesticides in one liter of water sample can be confirmed.
The obvious disadvantage of this approach is that it is not applicable to those 0.p.s
which d o not contain phenol or thiophenol moiety. As pointed out elsewhere,2s6an-
other disadvantage of this and similar procedures "of initially identifying a suspect
residue using a specific detector and then confirming using a nonspecific ECD after
hydrolysis, derivatization and clean-up may be viewed as a retrograde step, especially
since one of the advantages of specific detectors is to reduce initial sample clean-up."
However, the extraction step after hydrolysis and the column fractionation of the re-
action products used in this procedure provide additional substantiation of the iden-
tity. The extraction step removes all neutral electron-capturing compounds and the
column fractionation step groups the derivatives into appropriate fractions simplifying
subsequent ECD-GLC examination. These steps compensate for the use of a non-spe-
cific detector (ECD). Owing to the prominent sensitivity of ECD for PFB derivatives,
this approach is so far the only one suitable for confirming low levels of 0.p.s such as
those in water samples; clearly, the advantages outweigh the disadvantage.
Besides PFB ether derivative, a similar derivative (pentafluorobenzoate) has also
been used for the same purpose. Using thymol as a model compound, GC compari-
sionZS2of both pentafluorobenzyl derivatives show very little difference in detector
(ECD) response. Recently Bowman et reported a procedure for the confirmation
of an 0.p. (etrimfos) by reacting its hydrolysis product with pentafluorobenzoyl chlo-
ride to form a pentafluorobenzenzoate derivative. Unlike the PFB ether derivatives,
there are comparatively fewer actual investigations on pentafluorobenzenzoate deriv-
atives, particularly for the confirmation of 0.p. residues. The various derivatives used
in this approach for the identity confirmation of 0.p. residues are summarized in Table
25.
2. Derivatization of Intact 0.P.s

In comparing various approaches for chemical derivatization, it has been stated256
that "by far the most desirable approach is the generation of specific tests for deriva-
tization of the intact insecticides." This statement is true only when the other criteria
(e.g., sensitivity, background, interference, etc.) for a useful chemical confirmatory
test are equally met (see Section V on Criteria). For example, if a chemical derivatiza-
tion procedure for intact pesticide has high reaction background, narrow scope of
application, low sensitivity, it is obviously less desirable than a procedure involving a
pesticide hydrolytic product if this procedure has a lower reagent background, wider
scope of application, and more sensitivity. In comparing the desirability of an ap-
proach or a procedure, one has to consider all the key criteria for a useful confirmatory
test.
For the confirmation of 0.p.s there are several procedures in the literature that are
based on derivatization of the parent compounds. These procedures are summarized
in Table 26.
So far, the published procedures involve (1) oxidation of the parent 0.p.s to the
oxygen analogs (Singh and L a p ~ i n t ' ~ " )(2)
, CrCl, reduction (Forbes et (3) alkyl-
ation,260.261 and (4) trifluoroacetylation.262UV radiation263was also used, but up to
now its applicability is only demonstrated on one o.p., crufomnate. Under 30 min UV
irradiation in hexane, crufomnate is dechlorinated t o a deschloro derivative. Since the
number of common 0.p.s having chlorine moiety similiar to crufomnate is very limited,
the potential scope of application for this UV dechlorination procedure is obviously
also very limited.
a. Trifluoroacetylation
Since the report of Lau and M a r ~ m i l l e r ~in~ '1970 for the analysis of Landrin (a
mixture of two isomeric N-methylcarbamates) by reaction with trifluoroacetic anhy-
dride to form the N-trifluoroacetyl derivatives, reaction of trifluoroacetic anhydride264-
277 With carbamateS,264.265.268.270-273.277 urea,266.269.274-276and an o.p.262.264
having a sulf-
oxide or an active NH moiety have caught the fancy of many researchers. Out of this
large number of papers, only two papers deal with its application262or potential appli-
cation264for the confirmation of 0.p.s. Lawrence and M ~ L e o d ~report ~ ' in detail a
procedure for the analysis and confirmation of an o.p., AzodrinO [(3-dimethoxy-
phosphiny1)-N-methyl-ci+crotonamide] by trifluoroacetylation. This derivatization
technique has been demonstrated to confirm AzodrinB residues in strawberries at lev-
els as low as 1 to 2 ppb. It is a specific procedure for a specific purpose. Therefore,
its expansion to other 0.p.s with similar structures remains to be investigated. In con-
trast, Greenhalgh, King, and M a r ~ h a l applied
l ~ ~ ~ the trifluoroacetylation reaction to 4
o r 5 0.p.s and metabolites including DasanitB together with aldicarb (a carbamate)
and the previously investigated272Mesurol@ (also a carbamate). Although the title264
may suggest that it contains an analytical and confirmation procedure (depending on
one's intepretation), this paper emphasizes the chemical and theoretical aspects (struc-
tural elucidation by MS, NMR, and mechanistic interpretation) rather than the prac-
tical aspect as in the approach of Lawrence and M ~ L e o d However, . ~ ~ ~ an example on
the application for dasanit residue in soil at 1.1 ppm is given. Therefore, the practical
application of trifluoroacetylation for the confirmation of these 0.p.s in real life situ-
ations remains to be demonstrated.
Unlike dasanit and similar 0.p.s investigated, the sulfoxides of phorate and
Counter@ (not "phorate and Counter s ~ l f o x i d e " ~were ~ ~ )converted by the TFA pro-
cedure to the corresponding oxons at room temperature of 100°C after 15 min of
reaction. Although not mentioned in the text,264sulfoxide of demeton-SMe[(MeO)2
P0.S. CH2CH2.S0.Et]was observed278previously to be reduced to the sulfide analog
on heating and on storage. Another sulfoxide of similar structure [(Et0)2PS.0.C6H,-
4-SO.Me] was also o b ~ e r v e dto~ be ~ ~converted
. ~ ~ ~ to the corresponding sulfide under
the influence of heat. Therefore, thermal reduction at 100°C of oxydemeton methyl
(i.e., sulfoxide of dimeton-SMe) by Greenhalgh et al.264 to demeton-SMe
[(Me0)2P0.S.CH2CH2.SEt],is not unexpected. The demeton-SMe cannot be further
rearranged under heat without being broken down into a smaller molecule because it
is the end product of thermal i ~ o m e r i z a t i o n ~ ~ ~of. ~ ~demeton- ' - ~ ~ ~ OMe
[(Me0)2PS.0.CH2CH2.S.Et].This thermal isomerization has been proposed earlier to
occur via a cyclic intermediate.281-283 In general, dialkyl /3-ethylthioethyl and diethyl P-
ethylaminoethyl phosphorothionates were o b ~ e r v e d to ~ ~undergo
~ . ~ ~ similar
~ thermal
isomerization in good yield even at moderate temperature to the corresponding S/3-
ethylthioethyl and S/3-ethylaminoethyl isomers. Similar thermal isomerization is not
confined to these 0.p.s; for example, methyl parathion is isomerized 91% into S
methyl isomer by heating the pure crystalline compound at 150°C for 6.5 hr,283bal-
though the above-mentioned 0.p.s more readily undergo such isomerization. Eto283b
also mentioned that Lewis acids can accelerate this thiono-thiolo isomerization at low
temperature (see also Reference 284). In view of the above examples, the reaction of
phorate s ~ l f o x i d ewith
~ ~ TFAA
~ at room temperature or at 100°C could be conceived
t o involve ( l ) sulfoxide reduction to the sulfide and (2) thermal isomerization, probably
via a cyclic intermediate, to the oxon under the catalytic effect of TFAA with or with-
out heat. It is not known whether it is a stepwise or a concerted reaction. The above
discussion is summarized in the following scheme:
0 0 0
II II II
(MeO), PSCH, CH, SEt (MeO), PSCH, CH, SEt
Volume I 139
S 0 0
II II A I1
( M e O ) , POCH, C H , S E t (acid-eO),PSCH,CH2SEt
S 0 0
I/ \I 1I
( E t O ) , PSCH, S E t ------+
( E t O ) , PSCH, SEt
Based on the above discussion, the observed t r a n s f o r m a t i ~ nof~ ~

phorate
~ sulfoxide
and oxydemeton methyl (sulfoxide of dimeton-SMe) to oxons does not specifically
result from trifluoracetylation, rather it is due to the intrinsic characteristic and ease
of these compounds to undergo this thermal or acid-catalyzed transformation. The
term Counter sulfoxide is used in the text,264but its structure is not given although
structure of other compounds are 1 i ~ t e d . Counter@
l~~ is also known as Terbufos. Its
chemical name is S[(tert-butylthio)methyl]-0,Odiethyl phosphorodithioate. For
those who are just interested in the reaction conditions and reaction scheme of trifluo-
roacetylation on the investigated264pesticides, Cochrane's review2" nicely and clearly
summarized this in a paragraph and a table.
Greenhalgh et al.264also pointed out that "most pesticides with a sulfoxide moiety
react readily with TFAA under mild conditions (rt/15 min.) to form a single product
. . . ." However, "the presence of an NH group in the same molecule necessitated
forcing conditions (100°C) . . . to form the di-TFA derivative in order to obtain a
single product." This indicates that trifluoroacetylation is applicable only to those
0.p.s with sulfoxide or an active N H moiety.
Although trifluoroacetylation has a wide scope of application for many carbamate
and urea p e s t i ~ i d e s ~ ~ reacting
~ . ~ " ~ -with
~ ~ ' the NH moieties in these compounds, it has
a much narrower scope of application for 0.p.s since the number of commonly used
0.p.s having a N H or a sulfoxide moiety is considerably limited. However, it could be
demonstrated as a useful reaction for the sulfoxide metabolites of some 0.p.s; more-
over, as Lawrence and McLeod2'j2demonstrated earlier and was pointed out by Coch-
rane,2" "trifluoroacetylation of azodrin was the only feasible approach to its confir-
mation in strawberries after unsuccessful attempts at on-column and solution
alkylation." Therefore, trifluoroacetyIation could be a useful reaction under certain
circumstance for 0.p. confirmation.
b. Methylation by NaH/CH,I/DMSO System

Similar comments can be made on the use of the alkylation p r o ~ e d u r e using~~~.~~~
NaH/CH,I/DMS04 system for the confirmation of o.p.s, in that only those 0.p.s
containing active hydrogen (N-H) in the molecules can react. Thus, among the differ-
ent classes of o.p.s, this alkylation procedure has potential application only to phos-
phoramidothioates
and phosphoramidates
and there are only a few commonly used 0.p.s in these two classes. Therefore, the
general practical applicability of this procedure for 0.p.s confirmation is quite limited.
On the other hand, since many carbamates, urea, and triazine pesticides contain NH
moieties, this alkylation procedure has potentially a much larger scope of applicability
to these pesticides. In fact, it has been demonstrated by L a w r e n ~ e "that ~ this alkylation
system gave high yield and good reproducibility of quantitative measurement of these
compounds at residue levels (also see discussions in Volume 111, Chapter 1).
For the confirmation of o.p.s, however, the sensitivity of the alkylation procedure
is not sufficient for natural water samples, due to very high GLC background from
the reagents or reaction by-products. ECD cannot be used because of these excessive
electron-capturing background materials. Even using the specific detectors such as
AFID or FPD for examination of the derivatives, there is still considerable GLC back-
ground. This reagent background has been emphasized repeatedly by Cochrane in his
two reviews on chemical d e r i v a t i z a t i ~ n . ~ "In, ~a~study
~ by Lawrence and I v e r ~ o n , ' ~ '
this alkylation technique was "compared with silylation as a means of confirming two
diazinon metabolites in dog urine at 1 ppm level using a CCD (Coulson conductivity
detector) in the N mode."z55 Based on their finding,16' Cochrane commented that "in
general, silylation created less GC background than alkylation and the detector sensi-
tivity of the silyl derivatives was greater than that of the alkylated products." In this
case, BSTFAZ6'gave single products whereas alkylation (NaH/CH,I/DMSO) gave two
products f o r e a c h of the two diazine metabolites studied. The comment on lower sen-
sitivity and higher background of this alkylation procedure was repeated 3 years later
in the second review2" which states that "silylation with BSTFA261gave less GC back-
ground and better sensitivity than the a l k y l a t i ~ n ~method." ~ ~ . ~ ~ ' However, it should
be pointed out that the two diazinon metabolites [(2-isopropyl-4-methyl-6-hydroxypyr-
imidine and 2-(l'-hydroxy-l'-methyl)-ethyl-4-methyl-6-hydroxypyrimidine)] each con-
tain one or two hydroxy moieties as well as an active NH moiety. Also, Lawrence and
IversonZ6'carried out the reaction at room termperature rather than at 50°C as de-
scribed by Greenhalgh and K o v a c i c ~ v aThe . ~ ~pyrimidyl
~ O H and NH moieties are not
simultaneously m e t h ~ l a t e d , which
~ ~ ' results in two products from each metabolite. On
the other hand, silylation by BSTFA (N,Obis-(trimethylsily1)-trifluoroacetamide)at
room temperature gives a single product from each metabolite. Even if detector re-
sponse of the silyl and methyl derivatives is similar, the alkylation procedure generating
two products from each metabolite would have lower sensitivity. In fact, from Table
1 of Reference 261, even the combined responses (peak heights) of the two alkylated
derivatives is less than half of the response from the corresponding silyl derivative.
This shows that at least for these two diazinon m e t a b o l i t e ~ , ~alkylation
~' is consider-
ably less sensitive than silylation.
In addition t o the lower sensitivity due to the intrinsic detector response of the
methyl derivatives, the alkylation procedure using the NaH/CH,I/DMSO system at
room temperaturez6' shows a much higher reagent or reaction background. This will
decrease specificity as well as sensitivity of the procedure. It might be expected that
the reaction background would be even higher using the condition (i.e., reaction at
50°C) described by Greenhalgh and K o v a c i c ~ v abecause , ~ ~ ~ at higher temperature, for
some reactions such as this one, there is more possibility of interreaction among re-
agents, impurities, and sample CO-extractivesunless the reaction is of "destructive"
nature (such as concentrated H2S04and LAH reactions described earlier). In the latter
cases, the higher temperature will destroy more sample coextractives rather than in-
creasing reagent background.
In summary, as pointed out by Buchert and L ~ k k e "N-methylation , ~ ~ ~ used for sub-
stituted amide-H, performed by means of a proton-extracting base (e.g., t-BuOK or
NaH) and iodomethane as methyl donor is often used for making derivatives of pep-
Volume I 141
tidesHz8as reported in 1973 by Tanaka and Wien.245Since then, similar approach for
N-methylation has been investigated by many authors260~261~285-288for the confirmation
of a few 0.p.s and for the analysis of many carbamate, urea, and triazine pesticides.
Thus its wide scope of application t o several different classes of pesticide has been
demonstrated.
c. Chromous Chloride Reduction

Since the representation of a report entitled "Some Reactions of CrC12 on Organo-
chlorine Pesticides and their Utilization in Chemical Confirmative Tests" in 1970,"
Chau and colleagues16~"~90~120~121-123~150~151~203
have studied the reduction of CrCl, to
o.c.s, particularly on its application to chemical confirmatory procedures. Recently,
the usefulness of CrC12 reduction technique for confirmation purposes was independ-
ently demonstrated by the confirmation of mirex in environmental samples by Lusby
and in 1979 and Chau et a1.194.203 in 1978. All these investigations are on 0.c.s.
Inspired by the successful application of CrC12 on 0.c.s and particularly on 0.p.s con-
taining NO2 moiety,290Lawrence et al.289applied the CrC12 reduction to the confirma-
tion of some 13 NO2-containing herbicides and fungicides. However, M. A. Forbes of
the Water Quality Branch, Department of the Environment, Canada was the first per-
son t o apply the CrC1, technique in 1973290for the confirmation of NO,-containing
0.p.s (parathion, methyl parathion, and fenitrothion). Later, in cooperation with other
workers, a reportzs9 was published to extend the investigation to a few other 0.p.s
containing NO2 or CN moiety. The CrC1, successfully reduces the NO, to an amine
group in parathion, fenitrothion, and EPN. However, fenitro oxon, paraoxon, and
SurecideO decomposed during reduction with the described procedure and serecide
was not reduced.
Although Zn dust/HCl reagent can reduce all these 0.p.s and thus appears to have
a wider application than CrCl, reduction, this reagent gives much poorer yield of the
corresponding amine and produces several by-products; accordingly subsequent inter-
pretation of gas chromatograms becomes difficult. This undesirable effect of using Zn
dust in mineral acids has previously been discussed and compared with CrCl, reduction
in the reduction of several o . ~ . s ~ ~ . ~ ~ . ~ ~ . ~ ~ ~ . ' ~ ~
The number of commonly used 0.p.s having aryl nitro or aryl cyano groups is very
limited. Also, as mentioned above, not all 0.p.s containing these groups are reduced
by CrC1, by the described procedure. Thus, this CrC12 procedure has a very limited
scope of applicability. In addition, the reduction product of fenitrothion (aminofeni-
trothion) has a similar retention time to the reduction product of parathion (amino
parathion) on at least two commonly used GLC columns: 4% OV-17/6% QF-l and
4% OV-101/6% OV-210117columns. As pointed out by Ripley et al.291one problem
arising from this is that "if both these parent pesticides were present in the sample,
confirmation of the two might be difficult." Therefore, another column needs to be
investigated and used for the examination of the reaction products. This adds incon-
venience in applying this confirmatory test. Also, the amino derivatives of these three
o.p.s, particularly the aminofenitrothion, are not stable. However, disregarding the
disadvantages mentioned above, this procedure has two significant merits. Its lack of
general application also attributes to its specificity. Recently, in Eastern Canada feni-
trothion has been used in forest spraying to control spruce budworm (see Volume 11,
Chapter 2). The chromous chloride procedure is a simple, rapid and somewhat specific
method for its confirmatio\although the analyst must be aware of the limitations
associated with the procedure. Also, as experienced by this author and reported else-
where on the use of CrC12,15.16,17.182.195
the reagent as received has very low background
interference and the solution can be easily purified by shaking with hexane or benzene
under N2 atmospheres. Thus, the reaction extract can be evaporated to a low volume
(1 m1 or less) without significant contribution from the reagent. This is an important

aspect in the analysis of trace organics in natural water and in some lake sediment.
Due to the lower ECD responses of the amino derivatives, AFID and AFD specific
detectors are recommended290for use.
d. Chemical Oxidation
Among the various approaches discussed above for the confirmation of intact o.p.s,
the oxidation procedure developed by Singh and L a p ~ i n t ehas. ~ ~perhaps
~ the largest
potential scope of application. These authors have already demonstrated its applica-
tion to six o.p.s, and there are many 0.p.s having the P=S moiety which could be
potentially oxidized to the respective P=O analogs by the neutralized sodium hypoch-
lorite solution used in this procedure. Unfortunately, some of these oxygen analogs
are less sensitive and more difficult to analyze than the corresponding parent 0.p.s.
(Volume 11, Chapter 2, Section VII). Also, oxons may be present naturally. Hence,
for environmental samples such as water, this approach may not have sufficient sensi-
tivity.
3. Conclusion
Among the various approaches discussed above, the most ideal approach for general
application is the approach of hydrolyzing the 0.p.s followed by derivatization of the
phenolic or thiophenolic products with PFBBr or other perfluorinated reagent. Due
to the sensitivity of this approach and acceptable reagent background, the PFB proce-
d ~ r eis ~ applicable ~ ~samples.The chromous chloride procedure is also rec-
~ ~ . to~water
ommended for spcific application, particularly for fenitrothion in water samples. As
yet, for water samples there is no suitable method which has a wide scope of general
application and suitable sensitivity for the confirmation of those 0.p.s without pheno-
lic or thiophenolic moiety.
B. Phenoxyalkanoic Acids
GLC analysis of herbicidal phenoxyalkanoic and carboxylic acids requires derivati-
zation of the parent compounds to more volatile derivatives. The derivatives most
commonly used for this purpose are alkyl esters, among which the methyl esters are
widely used (see discussions in References 291 to 298). However, many methyl esters
have very short retention times under the usual GLC conditions. Interferences are
often encountered from sample CO-extractiveswhich generally appear in this region of
the chromatogram. Also, several of the commonly used herbicide acids give methyl
esters having similar or identical retention times on many mixed or single phase GLC
columns. Therefore, confirmation of thier identity is necessary to avoid misinterpre-
tation. Transesterification of the alkyl esters, particularly from the methyl esters to
higher alkyl esters and vice versa, and bromination are the only methods so far pub-
lished for confirmation purposes. Since the higher alkyl esters have longer retention
times299the ester peaks of the acids will not be clumped so closely together. Thus
identification is easier and less misleading. Ironically the ester peaks are also broadened
under the same GLC conditions. This makes detection of these ester peaks at lower
concentrations difficult and less certain. In other words, the broadening of the ester
peaks decreases the sensitivity of the transesterification procedures so that levels de-
tectable by the methyl ester procedure may not be observed after transesterification to
a higher alkylester. This sacrifice of detection sensitivity limits the usefulness of tran-
sesterification procedure for low level confirmation.
Generally, the volatility and hence the retention time (peak broadening) increase
with the molecular weight or branching of the ester, particularly in an homologous
series.299Thus transesterification of methyl esters to propyl esters300is favored rather
than to branch esters or higher esters in an homologous series in order to minimize
Volume I 143
loss of sensitivity due to peak broadening. However, as mentioned e l ~ e w h e r e ~the ~~-'~~

retention times of the propyl esters of some common herbicidal acids are still too close
for unambiguous identification. Compared with butyl and ethyl esters299the propyl
ester appears to be the best compromise in terms of sensitivity and GC resolution for
a mixture of acid herbicides. Thus a mixture of five acid herbicide propyl esters
(MCPA, 2,4-D, 2,4,5-TP, 2,4,5-T and 2,4-DB) can be successfully separated on a
mixed phase GLC column after transesterification from the corresponding methyl es-
ters by H2S04/propanol reaction at 95 to 100°C for 5 min. However, the presence of
other acid herbicides or methyl esters will cause some overlapping. Nevertheless, Yip's
procedure is a simple, quick, and reasonably sensitive method for the confirmation of
these five acid herbicides. It must be realized that this transesterifican confirmation
procedure is designed for those analytical procedures which methylate the acids to the
methyl esters. For other analytical procedures using other esters such as the increas-
ingly popular PFB esters295-298.300.301 or the 2-chloroethyl esters,295.298.302.303
the transes-
terification procedure of Yip294is not readily applicable.
As mentioned earlier in this chapter, transesterification of methyl esters of a few
herbicidal acids to the corresponding 2-chloroethyl esters was found66to be feasible
with an overall 60% yield by SM derivatization technique (A1203/H2S04/2-chloro-
ethanol). Some refinements on the procedure are still required before it is ready for
practical application. As m e n t i ~ n e d , a~ mixture
~ ~ . ~ ~of~ 2-chloroethyl esters of nine
common herbicide acids can be easily resolved on a commonly used mixed phase GLC
column under normal GLC operational conditions. Therefore, in terms of resolution
as well as sensitivity, tranesterification to the 2-chloroethyl esters may be more desira-
ble. However, Yip's procedure may have a lower blank.
Another procedure305 for the confirmation of herbicidal acid also involves acid-cat-
alyzed transesterification. This procedure was designed for the confirmation of isooc-
tyl and propylene glycol butyl ether esters of 2,4-D, 2,4,5-T and 2,4,5-TP in blood
and urine by transesterification to the corresponding esters. These long chain esters
are some common forms of the herbicides in formulations used for spraying (see Vol-
ume 11, Chapter 3 on phenoxyalkanoic acid herbicides).
This procedure, as pointed out by the authors, is not suitable for quantitative mea-
surements since "there is not any accordance between the results of the quantitations
before and after derivativation." They attribute the lack of reproducibility to possible
loss during heat treatment or nonquantitative extraction from the acid layer. Neverthe-
less, due t o the similar transesterification conditions (80°C, 5 min) of this procedure304
to that of Yip,294p r o p a n 0 1 ~can
~ ~be used instead of methanol,304thus minimizing loss
of the esters due to volatility, since propyl esters are less volatile. One of the key aspects
of Peteghem and Heyndrickx's304 work is to show that these important long chain
esters can be simply and quickly hydrolyzed and transesterified, thus setting the ground
work for future investigation (such as using 2-chloroethanol or other alkyl alcohol in
place of methanol). The subjectiveness, straightforwardness, and clarity of this paper
is admirable. The readers know exactly the strength and limitation of the proposed
method.
The work of Thier305 on bromination of the methyl esters of MCPA, mecoprop
(MCPP), and MCPB has been mentioned by C o ~ h r a n e . 'Thus ~ ~ after the usual meth-
ylation, the methyl esters of these three acids are brominated with bromine containing
5 % iodine in glacial acetic acid at room temperature for 10 min to yield the correspond-
ing mono-2-bromo derivatives. According to Thier,305these "bromination conditions
are so chosen that each herbicide forms only one monobromo from the above-men-
tioned acids or dibromo derivative (from some hrphenyl carbamates) which is ther-
mally stable and allows both identification and quantitative determination." However,
neither author mentioned the earlier work of Gutenmann and L i ~ k , ~who " in 1963
also brominated MCP and MCPB acids before methylation (BF3/MeOH). These au-
thors306also used iodine as a catalyst during bromination but used carbon tetrachloride
instead of HOAc305as solvent. In this case, after bromination and methylation, MCP
and MCPB each generated two peaks "which may represent two of the possible three
dibromosubstitution Therefore the method by Thier305 is preferred.
However, either bromination procedure may not be applied to other acid herbicides
or their esters, particularly those with more than one chlorine substitution in the phenyl
ring (e.g., 2,4-D, 2,4,5-T, and similar herbicides). As learned from elementary organic
chemistry, this is due t o the chlorine substituents deactivating the aromatic nucleus (by
strong permanent inductive effect) so that the electron density of the entire aromatic
nucleus is low and attack by an electrophilic reagent is difficult.
In summary, the most promising general confirmation procedure is the transesteri-
fication approach. The bromination procedure of Their305 for MCP, MCPP, and
MCPB is not suitable for most applications because the procedure involves bromina-
tion of the methyl esters. As an analytical procedure for these three acids, methylation
is not a good choice for derivatization due t o the very low ECD responses. Therefore,
as early as 1964, Gutenmann and Lisk302 used 2-chloroethylation instead of methyl
ester for the analysis of MCP in soil t o enhance detection sensitivity. Subsequent to
the finding of Chau and Terry in 1975295and 1976296that PFB esters of MCPA and
MCPB are the most sensitive derivatives as compared to Zchloroethyl and methyl
esters for ECD determination, a sensitive analytical method for MCPA and MCPB in
natural water was developed in 1976.297These findings inspired Sattar et a1.300a,300b to
develop a MCPA procedure for soil and Cotterill in 1979 t o extend the application to
MCPB and mecoprop in soil in 1979.30LThus it appears that the PFB esters are the
preferred derivatives for these MCP type herbicides. Therefore, Thier's bromination
procedure for the confirmation of the methyl ester of the three MCP type herbicides
is not expected t o have general application.
C. Carbamates and Ureas

T o facilitate the following discussion, the significance, meaning, and application of
GLC-chemical derivatization technique (GLC-CD) is again briefly discussed.
Confirmation of residue identity by GLC-CD technique involves the comparison of
retention times on one or more given GLC stationary phases before and after chemical
derivatization. In a broader sense, the chemical derivatization technique can be applied
t o confirm the identity of a residue after initial analysis by a non-GLC method such
as TLC or H P L C followed by formation of derivatives and reexamination of them by
GLC, HPLC, o r other methods.
As pointed out by C o ~ h r a n e "the
, ~ ~ ~use of element specific GLC detectors, e.g.,
flame photometric detectors (FPD), thermionic (AFID) and Coulson electrolytic con-
ductivity (CCD) has not entirely eliminated the need for pesticide residue confirmation,
since the specificity of these detectors is only relative."
Furthermore, in spite of the usefulness of a GLC-mass spectrometry, as commented
by Elgar,27 "there is a need for fairly simple, routine tests that can be incorporated
into the analytical procedures that every residue chemist can use." This simple, routine
test is the GLC-CD technique. Indeed, the "EPA Analytical Quality Control Manual
for pesticide^"^^^ stated that this technique is "becoming increasingly important for
corroboration of residue identity."
From the above and previous discussion, it should be obvious that a chemical con-
firmatory test such as the GLC-CD technique requires that a suitable analytical pro-
cedure be first available for the compound in question so that the confirmatory test
can be used t o confirm its identity after initial analysis. In other words, these tests
must be incorporated into the analytical procedures that the residue chemist can use.
Volume I 145
Confirmatory tests are practical tools to be used in conjunction with analytical proce-
dures in routine analytical operation, rather than designed as a technique for theoreti-
cal or fundamental research. To develop a confirmatory test without the existence or
reference to a suitable analytical method is no better than to invent a gasoline engine
without the existence of gasoline.
As pointed out by many authors (e.g., see References 211 and 307) derivatives of
pesticides are prepared not for confirmatory purposes alone. They are also prepared
for various analytical purposes such as to increase detectability for TLC, to increase
volatility sensitivity and stability for GLC analysis, to improve chromatographic sep-
aration, and even to enhance selectivity of detection. Except for some applications of
HPLC, derivative formation for the above-mentioned purposes are not in the sense of
a chemical confirmatory test, as discussed earlier, although there are certain enhance-
ments of selectivity after derivatization. If the residues before derivatization are ana-
lyzed by HPLC and their derivatives subsequently prepared are reexamined by HPLC
or GLC or other suitable techniques, then the derivatization process can be regarded
as a chemical confirmatory test per se.
For a detailed discussion on the derivatization procedures used for the analysis of
carbamates and ureas, see Volume 111, Chapters 1 and 2.
As discussed in detail in Volume 111, Chapter 1 and pointed out by C o ~ h r a n ein~ ~ ~
his 1975 review on confirmation of pesticide, "many of the insecticidal carbamates
and ureas tend to break down on GC columns, many publications have appeared on
their quantitative analysis via thermally stable derivatives .... Although (these various
procedures are) not originally designed as confirmatory tests, nevertheless, they can
obviously be used as such. Either the compound is derivatized intact, ... or the urea
or carbamate is hydrolyzed and the intermediate amine or phenol further derivatized
with various reagents."2ss This nicely summarizes in a few sentences the overall ana-
lytical approach of carbamates and ureas. However, it is difficult to agree completely
with his statement that "although not originally designed as confirmatory tests, never-
theless, (these derivatization procedures) can obviously be used as such." Because,
since "many insecticidal carbamates and ureas tend to break down on GC columns,"
and since they often have low detector response, they must be converted to thermally
stable and also more detection responsive derivatives for initial analysis. Once a deriv-
ative is formed, the derivatizable moiety in the molecule is usually used up; conse-
quently, other derivatives cannot be formed from this derivative. For example, if a N-
methyl or N,N-dimethyl carbamate is analyzed via derivatization of the phenolic or
thiophenolic hydrolysis product by, say, pentafluorobenzylation, the pentafluoroben-
zyl (PFB) ester will not further react with chloracetylation, thiophosphorylation, sily-
lation, dichlorobenzene, sulfonylation, DNT/DNP, and vice versa as listed in Table 1
of Reference 255 on "confirmatory tests for ... carbamates." This is readily under-
stood since the active functional group (-OH or -SH) is already blocked (derivatized)
by a PFB group in our example, so that further transformation is not possible under
the conditions described for chloroacetylation, etc. B r ~ m i n a t i o n ~ ~ hiodination
nd are
exceptions since they are known to attack the aromatic ring rather than (and maybe
in some cases as well as) the already reacted -OH or -SH moiety. Similarly, for a N
phenyl carbamate or a urea type insecticide after hydrolysis, the amines generated are
derivatized for GLC analysis. Again, once the amine group is derivatized by, say, 2,4-
DNP, it cannot be derivatized again for confirmatory purpose by, e.g., DNT, DNP,
or pentafluorobenzylation and vice versa. Again, iodination and bromination are the
two exceptions for the same reason as discussed for phenol-generating carbamates.
When the carbamates and ureas are analyzed by derivatization of the intact mole-
cules as in alkylation, then those derivatization procedures (Table 125s)for phenol or
amine-generating compounds could be used in this case as confirmatory tests rather
than for quantitative analysis as originally intended. However, although analysis is

based on specific and different derivatives generated from the urea or carbamate pes-
ticides by derivatization of the intact parent compounds, confirmation after hydrolysis
and derivatization of the phenol or aniline products is less specific because the same
derivative can be derived from two different derivatives of different intact carbamates
or ureas. For example fenuron (a urea) and propham (a Nphenyl carbamate) or their
N-acetyl, N-methyl, Npenfluoroacetyl derivatives give the same aniline after hydroly-
sis. Similarly, linuron and diuron or derivatives of the intact compounds (e.g., meth-
ylated linuron and diuron) also generate the same aniline upon hydrolysis. Thus, com-
menting on the analysis for ureas, Buchert and LQkkeZ8' stated, "the transformation
into anilines has the disadvantage that specific determination of such phenylureas as
linuron and diuron, which contain the same substituted-phenyl moiety is not possible
without previous and time consuming separation." Recently, Glad et al.308also men-
tioned that "distinction between linuron and the one of its metabolites could not be
possible by N-methylation."
Therefore, the general approach of initially identifying a suspect residue using a
more specific derivatization procedure (e.g., Nmethylation) and then confirming using
a less specific derivatization procedure (derivatization after hydrolysis) may be viewed
as a retrograde step, especially since some hydrolytic products cannot be differentiated
for some pesticides as discussed above.
Alternately, HPLC is a possible analytical methodology for the initial analysis of
these heat-labile compounds. However, at the time (around 1975) when most of the
confirmation tests to be discussed below were published, HPLC procedures for the
analysis of intact (underivatized) carbamates and ureas in real samples were lacking.
Even now (1979) the only practical detector for HPLC analysis of intact carbamate
and urea pesticides is the UV detector. Since many sample CO-extractivesexhibit UV
absorptions at various UV ranges, interferences from these CO-extractivesare often
encountered. Although it is theoretically possible to analyze these compounds by
HPLS-UV and apply some of the derivatization techniques as chemical confirmation
purpose, this integration requires considerable investigation before practical applica-
tion could be possible.
Another alternative of potential application of the following derivatization proce-
dures as chemical confirmatory tests could be to split sample extract into two or more
portions to form different derivatives before analysis by GLC. Matching analytical
results and interpretation from using two or more derivatives for a pesticide can be
construed as confirmation of identity. However, this will cut down sensitivity of detec-
tion if the same extract is split up, and it will increase analytical time if a large amount
of sample is extracted. It may be pointed out that none of the above alternatives are
discussed in the cited reviews and papers claimed for the chemical confirmation of
carbamates and ureas.
So far we have discussed theoretical possibility; for actual reports on the chemical
confirmation of carbamate and urea pesticides, there appear to be very few in the
literature, although there are a considerable number of chemical derivatization proce-
dures proposed for the analysis of these compounds (see Volume 111, Chapter 1 for
detailed discussion and also Table 1 of Reference 255 and pages 126 to 128 of Refer-
ence 211). As discussed above, it is not theoretically possible to use many of these
reactions for confirmatory tests. Those that can be considered possible are based only
on knowledge of elementary chemistry. Actual data or detailed procedures are lacking.
Now we are going to examine the very few chemical derivatization procedures pro-
posed in the literature to be used for the confirmation of carbamates and ureas by
chemical derivatization.
For the chemical confirmation of several N-phenyl carbamate and urea pesticides,
the most thorough and detailed investigation appears to be the report of Lawrencezs5
in 1976. This work provides sufficient details and data for the reader to set up his
method and t o know how it is applied in practice. But most important of all, this
confirmatory procedure was integrated and discussed with an analytical procedure. It
is definitely not a case of inventing a gasoline engine without the existence of gasoline
or vice versa.
In this work, seven ureas and three Nphenyl carbamates are analyzed as their meth-
ylated derivatives formed by the NaH/CH31/DMS04 procedure described earlier.
Identities of these herbicides were confirmed by cleaning the aniline moiety with so-
dium methoxide at 110°C from 1 to 18 hr followed by GLC analysis of the anilines
(without further derivatization). An electrolytic conductivity detector in the nitrogen
mode was used for all analyses. This approach is best expressed in the following equa-
tion using linuron (a urea) as an example:
Unfortunately, for all the N,N-dimethyl ureas (diuron, fluometuron, monuron, flen-
uron, and chloroxuron) the cleavage step by MaOMe required 16 to 18 hr at 110°C
and the last three ureas only gave about 50% conversion; therefore, the procedure
causes some inconvenience in applying to N,hrdimethyl ureas due to their resistance
t o hydrolysis even at 110°C. Furthermore, although not mentioned in the text,z85if
the sample contains linuron and diuron or fenuron and propham this confirmatory
procedure could not differentiate either one of these pairs of pesticides, as pointed out
earlier. There are, however, two noticeable advantages. The first is that the gas chro-
matographic conditions are the same for initial measurement and for confirmation.
The second is that "confirming of the methylated herbicides directly in the final extract
avoids the necessity of extracting another sample just for c o n f i r m a t i ~ n . "This
~ ~ ~saves
considerable time, an important aspect in routine operation.
As discussed previously, the NaH/CH,I/DMS04 procedure was advocatedZ6Ofor the
confirmation of pesticides with reactive N H moieties including o.p.s, carbamates, tria-
zine, and urea herbicides. We have examined the usefulness and limitation of this
methylation procedure for the confirmation of 0.p.s. Now we shall discuss its merits
for confirming carbamates and ureas. The merits of using this procedure for the deter-
mination (not confirmation) of N-phenyl carbamate and urea pesticides as methylated
derivatives have been demonstrated by Lawrence and LaverZBB and Lawrence.zs5How-
ever, the manner in which to use this methylation procedure as a chemical confirma-
tory test for these compounds as advocated by Greenhalgh and Kovacicovaz60~287 is
never clearly discussed by these authors. Generally, a pesticide is initially identified
and analyzed intact or as a certain derivative. Then its identity is confirmed by some
suitable technique such as a chemical confirmatory test. In the case of analyzing car-
bamate and urea pesticides, direct gas chromatographic analysis is not yet a general
routine procedure at all. As C o ~ h r a n pointed
e ~ ~ ~ out since "they tend to break down
148 Analysis o f Pesticides i n Water
on GC columns, many publications have appeared on their quantitative analysis via

thermally stable derivatives." Lawrence and Laverm8also commented that "the direct
GC analysis of carbamate and urea herbicides has been carried out by a number of
authors. Although different columns and GLC conditions were used, these authors
generally agree that for good separations and reproducible analyses of such com-
pounds, operational parameters must be rigidly controlled. Unfortunately, when doing
sample analysis such restrictions often make direct determinations impossible due to
interferences which cannot be avoided by remaining within the required GLC settings.
Many workers prefer to analyze these herbicides as GLC stable derivatives." Other
WOrkerS255.267-269.286,289.309
also expressed the difficulties of direct analysis due to ther-
mal instability of these compounds on GLC columns and some286.269 could not even
repeat earlier work of direct GLC analysis (see detailed discussion in Volume 111,
Chapter 1). Greenhalgh and K o v a c i c ~ v aalso~ ~ expressed
~ a similar view by stating that
"carbamate insecticides, like the urea herbicides, are difficult to chromatograph di-
rectly by GC due to decomposition." It may be noted that most of these authors refer
to thermal instability of these pesticides based on pure standards. In the real life situ-
ation, presence of sample CO-extractives and repeat injections will make this decom-
position problem even worse, although there is some indication the sample extract will
deactivate the column, thus facilitating the direct GC analysis.
From the above discussions and references cited, it is obvious that direct GLC anal-
ysis for carbamate insecticides and urea herbicide is not a general routine application,
particularly in the presence of a variety of CO-extractivesencountered in a routine lab-
oratory. These CO-extractivesmay accumulate and deposit onto the GLC column and
aggravate the problem of thermal decomposition. Therefore, for determination, these
thermally unstable compounds are derivatized to thermally stable derivatives such as
methylation by the NaH/CH31/DMS0 procedure. If these thermally unstable com-
pounds are initially determined or analyzed by their methylated derivatives, how could
the same methylated procedure be used for their confirmation? Therefore, although it
is obvious that this methylation procedure has good practical application for the anal-
ysis of these carbamates and ureas, it is not obvious how these authorsz6"intend to
use it as "a confirmatory test" such as for the two Nmethyl carbamates and the four
urea herbicides investigated. With one exception, these authorsz6" did not illustrate
how this N-methylation procedure was applied to these compounds. The exception is
the illustration on the "confirmation" of fenuron standard before and after methyla-
tion. Before methylation, the GC peak of 63 ng fenuron standard was no more than a
bump (Figure 3 of Reference 260) which could be obscured by sample CO-extractives.
On the other hand, the methylated derivative (1 1 ng) gave a sharp peak approximately
nine times higher in peak height than the underivatized fenuron. Another paper by the
same on the "confirmation of atriazine and fenuron by alkylation at ppm
level" shows no peak was observed from fenuron. The authors commented that "with
the GC conditions employed, fenuron could not be chromatographed." The authors
did not discuss how fenuron could be analyzed initially or how to apply their procedure
for confirmation purposes. Confirmation tests provide corroboration to the identity
of a pesticide from initial analysis. Thus, as discussed previously, they are used to
supplement analytical determination. Therefore, a confirmatory test must be inte-
grated to one or more analytical procedure before it can have served its purpose.
Similarly, trifluoroacetylation (see previous discussion on 0.p.s and Volume 111,
Chapter 1) for derivatization of carbamates is more appropriate to be regarded as an
analytical approach rather than as confirmatory tests as Greenhalgh et al.264proposed.
Particularly for sulfoxide-containing carbamates such as mesurol sulfoxide and sul-
fone, as Greenhalgh et al.272pointed out, these compounds "like most Nmethyl car-
bamates have poor GC characteristics and must be derivatized for analysis." However,
confirmation by trifluoroacetylation has been documented309for ethyl carbamate (not
a pesticide) but this is a special case since ethyl carbamate can be reproducibly pyro-
lyzed at 800°C and detected by Coulson electrolytic conductivity detector. This tech-
nique could not be readily applicable to carbamate and urea pesticides, particularly
the lack of s e n s i ~ i t i t y(0.1
~~~ . ~ ~and
ppm) ~ unpredictable thermal decomposition which
preclude the general routine application by this approach. Even if applicable, this ap-
proach requires additional sample extracts and the setting up of different instruments
just for confirmation purposes.
Recently, in 1979, Hall and Harris310 investigated the direct gas chromatographic
determination of 32 carbamate pesticides (not urea pesticides) applying a highly deac-
tivated GLC column (CarbowaxO 20M-modified supports) and electrolytic conductiv-
ity detector. Some of the carbamates were investigated in the presence of soil extract.
This report shows some promise in that this approach could be used for initial deter-
mination of underivatized carbamates. However, there are still some problems to be
ironed out. For example, poor results were obtained for a few carbamates such as
aldicarb, methomyl, and others. Some decomposition was also indicated for carbaryl,
methiocarb, and Meobal. The lowest detection limit was generally at the 0.1 ppm level
for the 22 carbamates fortified in soil, but carbaryl gave insufficient response at this
level to allow determination. If in the future this approach can be demonstrated to be
used as a general routine analytical procedure for the analysis carbamates, then pro-
cedures such as N-methylation or trifluoroacetylation currently used for the derivati-
zation and subsequent analysis of the intact carbamate pesticide could be utilized as
chemical confirmatory tests for these compounds as advocated in 1975.211.260.288
D. Triazines
1. General Discussion
Atrazine is the most widely used herbicide in North America. (see Volume 111, Chap-
ter 3 for discussion on analytical methodology and structures of some commonly used
triazines). Its basic molecular structure is the symmetrical triazine ring. Several com-
pounds with this basic structure are also used as herbicides, particularly those contain-
ing a 2-chloro moiety as in atrazine. Therefore, these compounds are also referred to
as 2-chloro-striazine herbicides. The basic molecular structure of 2-chloro-striazine
is depicted below:
The three nitrogen atoms in the triazine ring in these compounds are symetrically dis-
tributed and hence the symbol ''Pis added to the nomenclature t o differentiate then
from 1,2,4-triazine (asym-triazine) or 1,2,3-triazine (v-triazine). Triazine herbicide
with these nonsymmetrical nitrogen rings is much less common. Sencor is one of the
few examples of asym-triazines used as herbicide.
Another major type of striazine herbicides is the 2-thiomethyl-striazines, such as
prometryne, ametryne, and metoprotryne.
The 2-chloro and the less commonly used 2-thiomethyl-symmetrical triazines are
generally used as herbicides for post- or pre-emergence (sometimes both) control of
weeds in corn, potatoes, and other crops. Dyrene (2,4-dichloro-6-(-0chloroanilino)-
striazine) however is used as fungicide.
Analysis of parent triazine herbicides are generally amiable to GLC analysis partic-
ularly with N-specific detector (see Volume 111, Chapter 3 and also Reference 311).
Therefore, chemical derivatization procedures are used for confirmation of their iden-
tity after initial determination. Since almost all the published work is on the chemical
confirmation procedures of atrazine and similar compounds (i.e., 2-chloro-striazines),
the following discussions will emphasize on these compounds.
2. Confirmation Procedure for the Parent Triazines

At the present time (July, 1979) there appear to be only five papers260~312a~313~268~320
and four methods that deal with chemical confirmation of the parent compounds. The
first one is that of Wales and M e n d o ~ a , who
~ ~ ~reported
" in 1970 on confirmation of
Dyrene, an striazine fungicide. These authors reacted Dyrene with methanolic NaOH
at room temperature for its confirmation in plant extracts. Later3lZbheating with meth-
anol at 65°C without NaOH was found sufficient. The reaction is summarized by the
structure below:
It may be pointed out that the structure of Dyrene was purposely drawn in such a
manner to emphasize the symmetry of the triazine moiety and hence the chemical
equivalence of the two chloro substitutions at C-2 and C-4.
Since the nitrogen atoms (C-N=C) in the triazine ring are more electronegative than
the methine carbon, they exert an electron attractive effect on the n-orbital system in
the triazine ring causing the carbon atoms n-deficient (i.e., having a fractional positive
charges). Thus the carbon is susceptible to nucleophilic attack by nucleophiles. There-
fore the Cl-C bond in 2-chloro-striazine is easily replaced by methoxy ion. In the
case of Dyrene, the echloroanilinic substitution at C-6 of the triazine ring, probably
because of both resonance and inductive effects, even make the chlorine atom at C-2
or C-4 more labile and easily replaced by nucleophile. Therefore, Dyrene can be readily
(20 min) converted at mild temperature (65OC) to Zmethoxydyrene in methanol (a
weak nucleophile) without the need for alkaline as in the case of methoxylation of
other 2-chloro-striazines. Longer reaction time will form the dimethoxy derivatives as
shown above.
Reaction of Dyrene with ehanol, propanol, and butan01~'~ under the same conditions
is very slow. As expected, in the presence of alkaline (NaOH or NaOMe), Dyrene is
readily converted even at 25°C to the monomethoxy derivative in 5 min or less. The
reaction is faster using NaOMe.
Due t o the expected ease of converting 2-chloro-striazines to 2-methoxy-striazines
based on the chemistry and Mendoza's work discussed above, methoxylation is a logi-
cal approach to be investigated for the confirmation of other 2-chloro-striazines.
Thus, using similar methoxylation procedure to that employed by Mendoza et
Lawrence313 investigated the application of this methoxylation technique to three 2-
chloro-striazines (atrazine, propazine, simazine) and found that the conversions to
the respective Zmethoxy derivatives were quantitative. Stirred on a vortex mixer for 1
min with 25% sodium methoxide/methanol solution, atrazine was converted to atra-
tone, propazine t o prometone, and simazine to simetone. Except for atrazine, unfor-
tunately the detector (Coulson conductivity) responses of the derivatives were not com-
pared with those of the parent compounds. However, based on the chromatogram of
atrazine before and after methoxylation (Figure 4 of Reference 313), the methoxy de-
rivative (atrazone) has a higher detector response. It may be pointed out that Wanless
and Mendoza3I2 found that the mono-methoxy and di-methoxy derivatives of Dyrene,
as expected, have very low ECD responses. Similarly, the ECD responses of the three
methoxy triazines in Lawrence's work are also expected to have very low ECD re-
sponses as compared to the parent compounds. Lawrence313also studied methylation
of these three 2-chloro-striazines, sencor, and prometone using the NaH/CH31/
DMSO, procedure reported in 1973 by Cochrane and Greenhalgh,314who also studied
the methylation of atrazine (Figure 2 of Reference 256) and atrazone. This is the same
alkylation procedure also reported by Greenhalgh and K o v a c i c ~ v ain~ 1975
~ ~ and dis-
cussed earlier. Lawrence confirmed the derivatives to be a dimethylated product, and
based on the study of sencor, simazine, atrazine, propazine, and prometone proposed
the general scheme for this alkylation as follows:
The alkylated sencor was found3I3by mass spectrometry to be
Unfortunately, as in the case for methoxylation, Lawrence313only used atrazine to

compare the detector (CCD) response before and after derivatization. In this case,
methylated atazine has a similar detector response to the parent compound. It is also
of practical interest to know whether the other methoxylated and alkylated triazines
studied have a higher, lower, or similar detector response as the parent compounds.
Greenhalgh and K o v a c i c ~ v areported
~ ~ ~ an alkylation procedure exactly the same as
that of Lawrence313except for the minor difference on the volume of DMSO used (0.1
m1 instead of 0.5 mf3I3)and reaction at 50°C for 10 min instead of 45°C for 10 min.
With the exception of prometryne (a 2-methoxy-striazine), these authorsZ6Oalso stud-
ied the same triazines (atrazine, atratone, and prometone) as Lawrence3I3for the con-
firmation of their identity by NaH/CH31/DMS04. However, instead of a conductivity
detector, they used an AFID. Except for atrazine, unfortunately, there are also no
chromatograms or discussion to indicate the detector response of the parent com-
pounds and of the methylated derivatives. Therefore, it is difficult to assess the prac-
tical sensitivity of this procedure. In the same year, these authors reiterated288previous
results260on the methylation of atrazine and fenuron in a short publication which also
shows a chromatogram of atrazine before and after methylation (alkylation). How-
ever, the chromatographic tracing show a somewhat larger increase of detector (AFID)
response for the methylated atrazine as previously noted.260In the same year, this
alkylation of atrazine was again described in another report3', but this time the prac-
tical application of this reaction together with silylation and diazomethane methylation
for the confirmation and analysis of atrazine and hydroxyatrazine in soil were fully
discussed. The conditions for the alkylation procedure are somewhat different from
those p r o c e d ~ r e s ~mentioned
~ ~ . ~ ' ~ earlier in that 20 mg NaH (instead of 10 mg260.313)
was used and the reaction temperature was raised to 70°C and prolonged to 1 hr. The
higher temperature and longer reaction time (and perhaps also the larger amount of
NaH) are necessary to react the more resistant 2-hydroxy atrazine. Since the alkylation
o f atrazine by NaH/CH,I/DMSO was repeatedly reported by different or the same
authors under similar or different conditions to obtain quantitative conversion313
"86% yield (assuming the AFID response to be the same as for a t r a ~ i n e ) "and
~ ~ "90%
~
yield",31s there is no doubt that this conversation is a very high yield one.
In addition to methoxylation and alkylation, silylation was also investigated for the
confirmation of parent triazines since they contain NH moieties. However, it appears
atrazine is the only parent triazine studied for silylation. Cochranezs6mentioned three
groups of scientists who studied the BSFTA silylation of atrazine as a confirmatory
test (Figure 2 of Reference 256). However, we could only verify two of the references,
the work of Cochrane and Greenhalgh3I4(1973) and Kahn, Greenhalgh, and Coch-
rane.3's We could not locate the third reference (Reference 17 mentioned in the re-
viewzs6)on silylation of atrazine by Greenhalgh and Kovacicova, Journal o f Agricul-
tural Food Chemistry, stated as being in press at that time (1975). Silylation is not a
desirable approach for confirmation of atrazine because Khan et al. observed316that
"at low temperature (<120°C) no reaction took place" and requires 190°C to obtain
over 90% yield. Therefore it is not convenient to carry out routinely. As pointed out
by these authors, Coward and Smith3I6had discussed the undesirable effect of SiO,,
formed by combustion of silylating agent, causing a gradual loss of sensitivity of the
AFID. Therefore Coward and Smith "doubt the capacity of the detector to handle
continuously the numerous injections associated with rapid isothermal analyses." In
fact, Khan et observed at 40% decrease of AFID sensitivity after only ten consec-
utive injections of silylated material.
Another procedure for the confirmation of triazines was the two-step reactions also
described in the report by Lawrence313in 1974. As pointed out by this author, it was
based on the method of Cee and Gusparic317 in 1972 for the determination of the s
triazine ring in organic compound. In this procedure, the triazines were hydrolyzed to
release the amine(s) by acid (HC1) at 150°C in a stoppered tube for 6 to 18 hr. The
amines were then reacted with DNFB at 85OC (2,4-dinitrofluorobenzene) t o form the
dinitroderivative(s). Depending on the amino substituents of the 2-chloro-striazines
investigated, acid hydrolysis gives one or two alkyl amines which gives the same num-
ber of dinitroderivative. The reactions are summarized below:
l
OH
+ R,NH2 + R,NH, + HCI
The major disadvantage of this procedure is the length of reaction time (6 to 18 hr)
and the inconvenient reaction conditions (150°C). The other disadvantage is the high
background interference caused by the unreacted DNFB and it required an additional
step of heating the final reaction mixture with NaOH for 10 min to remove excess
DNFB. This step adds extra inconvenience to the already time-consuming procedure.
Volume 1 153
The other major disadvantage of the above procedure is the lack of specificity and
cannot easily confirm atrazine, propazine, and simazine when presented together be-
cause one of the amine is common to either two of the triazines. Interpretation of the
chromatogram would be difficult. Also, if the common metabolites, 2-methoxy or 2-
hydroxy derivatives are also present, distinction among the metabolites and the parent
triazine will be impossible. Interferences can also come from other triazines or metab-
olites which generate the same amine and hence the same DNFB derivatives. The above
situation can be summarized below:
Atrazine
Atratone (2-Me0 derivative) EtNH, + isopropylamine
1
2-Hydroxyatrazine (Me,CHNH,)
Ametryne
,
Simazine
Sirnetone (2-Me0 derivative) H+ EtNH,
2-Hydroxy simazine
Simetryne
l-
Propazine
Prometone (2-Me0 derivative) H+ Isopropylamine
2-Hydroxy propazine
Prometryne
In addition, the author3I3did not discuss the retention times of the DNFB derivatives
of ethyl- , isopropyl- , and methyl-derivatives generated from the investigated triazines.
They may or may not have sufficient resolution for unambiguous identification by the
GLC condition described.
3. Summary
In summary, so far, there are four procedures described in the literature for the
confirmation of parent triazine herbicides. The procedures are methoxylation, alkyla-
tion (NaH/CH,I/DMSO), silylation, and acid hydrolysis-DNFB reaction. Each pro-
cedure was investigated on one to four of the following triazines: atrazine, simazine,
propazine, sencor, or prometryne.
Lawrence3I3 investigated three reactions (NaH/CH,I/DMSO,, CH2N2,hydrolysis-
DNFB) on some of the seven triazines and 2-methoxy metabolites listed, but provided
specific data only on atrazine. Except in the methylation reaction, some data on five
traizines were presented. By comparing these reactions for confirmation it was stated313
that "for confirmation of chloro-striazines, the methylation reaction with methyl io-
dide would be preferred. The hydrolysis-DNFB reaction was found useful for further
characterization of the triazines by determination of the ring amino groups." The au-
thor also suggested that the above reactions be used in a stepwise manner. Unfortu-
nately, there are very little actual data presented to compare these three reactions on
sensitivity and GLC resolution of the derivatives of the seven mentioned triazines and
metabolites under the same experimental conditions. Based on Figures 3 and 4 of this
reporL313it appears that the methoxylation procedure for atrazine has a slightly better
sensitivity of detection than that of the methylation procedure when the peak height
of each of the derivatives is compared respectively with that of atrazine in these two
chromatographic tracings, assuming that in each tracing the GLC conditions remain
the same for the parent compound and the derivative. Since simazine instead of atra-
zine was used for illustration of the acid-DNFB procedure (Figure 5 of Reference 313),
we cannot compare its sensitivity with the other two procedures. However, one could
assume all the procedures have similar sensitivity since "these derivatization reactions
were applied to residue analysis at concentrations of less than 0.05 This as-
sumption, of course, is valid only when the sample size and volume of sample extract
are the same in these investigations.
Kahn et investigated three reactions for the analysis and confirmation of atra-
zine and its 2-hydroxy metabolite in soil. Only ~ i l y l a t i o n and
~ ' ~ alkylation with CH31
are applicable t o atrazine. Methylation with CHzN,320is only suitable to the 2-hydroxy
derivative. Silylation is an undesirable reaction for AFID and atrazine requires high
temperature (190°C) for 90% conversion. Therefore, only the alkylation procedure is
suitable for atrazine under their conditions.
Based on the above discussion the best procedure for the confirmation of atrazine
and perhaps also other similar triazine herbicides is the alkylation precedure using the
NaH/CH31/DMS04 system. The alkylated (methylated) derivative of atrazine has
identical (1.0),313slightly higher (1.05),260 and somewhat higher (1.2)288detector re-
sponses as compared to that of the same concentration of atrazine before and after
reaction, using CCD in the first case and AFID in the last two cases. The values in
parentheses are estimated by calculating the peak heights of atrazine and its methylated
derivative from the chromatographic tracings in these publications. This alkylation
(methylation) procedure is also much more specific than methoxylation and acid hy-
drolysis-DNFB reaction since different derivatives are obtained from different parent
triazines, and its Zhydroxy or Zmethoxy metabolites. However, it remains to be seen
whether the alkylated derivatives of several similar triazines and their common 2-meth-
oxy or Zhydroxy metabolites can be easily resolved under normal GLC conditions.
Lawrence313 showed methylated derivatives of sencor, simazine, and atrazine can be
resolved from methylated propazine and prometone on a 4% SE-30 column at 175°C.
The latter two derivatives were not resolved. The difference between them is the re-
spective Zchloro and Zmethoxy substituents.
It may be pointed out that there are several reaction^^^^-^^^ reported in the literature
on triazine compounds, but these are either not designed as confirmatory tests and
lack the necessary details and conditions for such application or the nature and condi-
tions of the reaction are so inconvenient that to perfom that application is impractical.
There is, however, one approach mentioned by CochraneZs6that could have some lim-
ited potential applicability. It is the hydrolysis of atrazine (or similar compounds) to
the corresponding 2-hydroxy derivative which could then be silylated or methylated
for GLC examination (Figure 2 of Reference 256). This approach is based partially
only on theoretical possibility. However, there are several known observations and
theoretical considerations that are discouraging. Besides being a two-step reaction, si-
lylation or methylation with CH2N, of Zhydroxy derivative is not convenient to per-
form. It requires heating314 at 190°C for good conversion to the silyl derivative and
prolonged reaction3I4 (3 hr) for CH2N2methylation. These reactions have several side
Of course, silylation also prevents the use of the most desirable detector
(AFID or similar N-specific detector) for triazine compounds. The conditions for hy-
drolysis of atrazine to the 2-hydroxy product may be more difficult to carry out in
practice than it appears because the right hydrolytic condition has to be found so that
the di- and tri-hydroxy313atrazine would not be formed in significant amounts.
4 . Confirmation o f Major Meta bolites o f Triazines

Since Zhydroxy analogs, the main metabolites of 2-chloro-triazines, "cannot be
readily gas chromatographed it is necessary to derivatize them in order that they may
be q ~ a n t i t a t e d . " ~Therefore,
'~ silylation, methylation, or P C l s procedure is used as
analytical procedure rather than as a confirmatory test. On the other hand, the 2-
methoxy triazines, also common metabolites, can readily be analyzed by GLC. Their
Volume 1 155
confirmation can be achieved by alkylation (methylation) with CH31of the NH moie-

ties. Thus p r o m e t ~ n e ~and
~ ~a,t~r a' ~
t ~ n e ~have
~ ' been more or less demonstrated to be
confirmed by the alkylation procedure.
5. Conclusion
In conclusion, methylation by CH31 is the only practical confirmation procedure for
triazines and their Zmethoxy metabolites. Although several authors studied a few of
these compounds, the actual application of the alkylation procedures to a mixture of
common triazines under real life situations has not been established.
V. OVERVIEW AND SELECTION CRITERIA OF CHEMICAL

DERIVATIZATION CONFIRMATION TECHNIQUES
In summary, chemical derivatization is no doubt a useful and cheap alternative to

the GC/MS in identity confirmation for routine pesticide residue analysis. It might be
emphasized that the scope of GC/MS is almost unlimited, at least in theory, in con-
firming a large number of volatile organic compounds whereas the chemical derivati-
zation technique is limited to those compounds for which procedures are available.
However, in a routine laboratory the main function is not to find out what possible
contaminants or pollutants are in the samples but to determine the presence or absence
and levels of certain known contaminants or pollutants from a list which is compiled
for compounds to be analyzed as required by government acts or regulations for mon-
itoring and surveillance purposes. These compounds are those that are toxic, widely
used in agriculture, known as industrial discharge, and so on. The objectives for anal-
ysis of these compounds are numerous: to ensure that the levels of pesticides do not
exceed certain limits in food or feed for safety human or animal consumption, to
obtain analytical data on existing pollutants in the environment for environment pro-
tection and pollution control purposes, to evalcate effectiveness of certain pollution
control techniques, and to monitor ecosystems for possible deterioration or improve-
ment as a result of man's activities.
The list of chemicals and substrates (sample type) to be monitored by a routine
analytical laboratory are governed by the term of reference and goals of a routine
analytical laboratory; for example, an environmental routine analytical laboratory will
be concerned with the analysis of environmental samples such as natural waters, sedi-
ment, and other waterborne substrates for a selected list of agricultural and industrial
chemicals that are known to affect the environment. The list is necessarily periodically
updated or modified based on other activities such as research results and government
legislation to reflect new situations and meet new requirements. Decidedly, the purpose
of a routine lab is not to attempt to find unknown or uncharacterized pollutants or
chemicals in a sample because it can be a very time-consuming endeavor and it requires
very sophisticated instrumentation, the most important one being a GC/MS with a
computer. To find out what possible pollutants are in a sample can take from several
months to more than a year. It defeats the primary function and objective of a routine
laboratory for it to engage in such an activity. This is a research area since for many
uncharacterized pollutants no analytical methodology is available. However, during
routine analysis some extraneous peaks in GLC may be observed and if they are sus-
pected to be of high level and occur frequently in samples an alert analyst should
inform the research people to find out what possible unknown pollutants are there.
A GC/MS with data system is a must for research-monitoring activities. On the
other hand, it could be an asset or a disadvantage in a routine laboratory. Since a GC/
MS with computerization is a sophisticated instrument it requires specialized personnel
to use it efficiently and maintain it effectively. It is an expensive and ineffective way
t o use the instrument in those routine laboratories which analyze a small list of com-
pounds (less than 40 to 50 compounds) and which only use the GC/MS primarily for
confirmation o f identity. Some of the reasons for this view are
1. Normal sample cleanup for residue analysis in some cases is not suitable for GC/
MS analysis and hence increases time of analysis. It requires additional cleanup for
GCIMS analysis and hence increases time of analysis. Also, due t o certain interfer-
ences, low level confirmation can be a problem in G U M S work.
2. In practice, most routine laboratories used multi-column techniques to substanti-
ate identity. This is for expedience. Extra confirmation is only performed in case of
doubt and only on one sample out of, for example, every 10 to IS samples as long as
they are from the same source or of the same type, because regardless of the confir-
mation technique employed, it is time consuming if confirmation is done on every
single sample. In practice, low level confirmation by G U M S for routine samples can
be very time consuming compared with other confirmation techniques. Moreover, due
t o the low levels of pesticides to be determined in the presence of considerably larger
amounts of CO-extractives, it is not necessarily as specific a technique in practice as
the theories may suggest. No routine laboratory which this author is aware of is using
this system for confirmation purposes on every single sample, because it is time con-
suming and expensive.
It is obvious then, if G U M S with data system is used only for confirmation pur-
poses, the cost and time to maintain this system is out of balance to its effective use.
Due t o the time and effort t o maintain the system in optimum condition for low level
confirmation, we have seen it becoming a burden to certain routine laboratories in the
sense that laboratoy efficiency and productivity suffer.
In a routine laboratory chemical derivatization techniques for confirmation pur-
poses offer a quick, sensitive, and effective alternative provided methods are available
for the compounds in question. This approach can be supplemented by TLC-GC, p-
value, and multi-column techniques.
If one choses chemical derivatization techniques as the primary approach for confir-
mation of identity, one should consider what the criteria are for a good chemical con-
firmation procedure.
Criteria for a useful chemical confirmation test have been briefly mentioned by
K a ~ f m a nin~his
~ Ph.D. thesis, by Cochrane in his descriptive literature review,255by
Cochrane and Mayburyzl" and by Greenhalgh and K o v a c i c ~ v aHowever,
.~~~ it appears
one or two criteria were not mentioned and there are also some misconceptions. The
enlarged and revised version o f the criteria of an ideal derivatization procedure for
confirmation are now listed and discussed in more detail below.
A. Sensitivity
The sensitivity for a chemical confirmatory procedure should be at least equal or
close to that of the method of determination. For 0.c.s residue analysis, invariably
GC/ECD is used for determination. In this case, the product produced by a confir-
matory procedure should have similar or better ECD response to that of the parent
pesticide. While for some food samples, a confirmatory test at milligram per kilogram
(ppm) level may be satisfactory, a confirmatory procedure is generally useless for most
water analysis if it cannot confirm ppt (1012)level.
B. Low Reagent Background

A chemical confirmation procedure with a low reagent background enables the re-
action extract to be concentrated to a low volume and hence increases the sensitivity
of the procedure. Even though the intrinsic detector response of the product could be
somewhat less satisfactory, a low reagent background can compensate this weakness
Volume I 157
to a certain extent. Also, low reagent background gives a "clean" chromatogram for
easy interpretation. It should be mentioned that some reagents are not available com-
mercially in pure form and their in-house purification is lengthy or difficult. Ob-
viously, procedures using these reagents are less desirable.
C. Ability t o Destroy Sample CO-Extractives

With only a few exceptions, most pesticide residue analyses and trace organic anal-
yses for environmental or other samples require cleanup of sample extracts before
determination such as by GC. However, no cleanup procedures such as liquid-liquid
partitioning, column, or TLC are 100% effective. At best, these steps minimize sample
CO-extractives.Therefore, a chemical confirmation procedure that has good ability to
destroy or further minimize any sample CO-extractivescan be a great advantage and
would have a larger scope of applicability to various sample types. On the other hand,
a chemical confirmation procedure that produces extraneous peaks either from reagent
impurity or from reaction by-products is therefore undesirable.
D. Inability t o Produce Detector-Responsive Products with Sample CO-Extractives

A derivatization procedure can react with sample CO-extractivesas well as the pesti-
cide in question. The reacted CO-extractivemay or may not have significant detector
responses. If these products are very volatile, have low or no detector response, or
become water soluble and removed during extraction, they will not show up in the
chromatogram. This becomes the situation in Criterion C. However, if these products
(from reacted CO-extractives)show significant detector responses in a chromatogram,
particularly in the areas of other commonly occurring pesticides, the procedure is not
satisfactory. The exact nature of CO-extractivesis often unknown and varies with sam-
ple type, and for some samples such as sediment and water even varies with sample
locations; therefore, a derivatization procedure is less desirable if by virtue of its
known mechanism or behavior it can react with several common types of compounds
to enhance the possibility of reacting with CO-extractives.Thus the chromatographic
patterns and degree of interferences are less predictable. UV photolysis and to a lesser
degree halogenation or perchorination procedures are notable examples for certain
samples.
E. Unique Retention Time of Derivative

Even if one is analyzing only a single pesticide or compound in a sample, other
pesticides or compounds may be present. The chemical confirmation procedure for
the compound in question should produce a derivative having a retention time different
from those retention times of other pesticides generally expected to be possibly present.
For specificity, it is even more desirable for the derivative to have a retention time well
removed from those areas of the commonly expected pesticides which do not form
derivatives or from those areas where other pesticides may form derivatives in this
reaction. This characteristic provides additional latitude on ruggedness to the proce-
dure.
F. Formation of One Single Derivative Peak*

Although it was suggested216that a "double (two derivative peaks) peak gives better
confirmation than a single peak when sensitivity is not critical" our experience indi-
cates that this often causes confusion in actual samples since peaks of other pesticides,
degradation products, o r CO-extractivesmay also exist. As mentioned previously for
o.c.s, column cleanup and fractionation (Table 5) is a commonly used step before
* Two or more peaks are generally confusing.

determination by GLC analysis. This column chromatographic process groups pesti-

cides into three or four fractions for GLC analysis. In one fraction, there are usually
several pesticides (see Table 6). If a chemical confirmation procedure for a pesticide
generates two or more derivative peaks, the chromatogram could be very confusing
with the presence of other pesticides or their derivatives in this fraction. Also, the
sensitivity of the derivatization procedure is often lower because two or more products
are formed from one parent compound. In unusual circumstances when the pesticide
in question is isolated in the absence of other related pesticides and CO-extractive,then
a derivatization procedure giving two derivative peaks could be of advantage over that
giving one derivative peak to provide additional confirmation, provided that other
factors such as sensitivity, reagent background, etc. are equal in these two procedures.
G . Formation of a Derivative Peak with Height Equal t o or Higherman the Parent

Compound*
In this aspect, there appears to be some misunderstanding on the differences between
C D - GLC procedure and synthetic chemistry. For example, one reportzo6stated that
"in order for a chemical reaction to be suitable as a confirmation test for a pesticide,
it should ... give single specific product in high yield at the residue level." Another
reportzs6also suggested that "a good yield" is one of the "criteria" for a useful chem-
ical confirmatory test." Our view and experience indicates that good yield is not a
necessary criterion provided the derivative peak has a good detector response. The
yield can be very low. This to me is the key consideration. For example, if the detector
response of derivative A from a reaction is ten times as great as that of derivative B,
then the required yield for A may be only 10% to obtain the same level of response as
derivative B with 100% yield. Consideration of the yield of a derivative is important
only under the following two situations. The first one is when comparing two proce-
dures producing the same product. Obviously, the procedure giving a higher yield of
the derivative is better than the other procedure, provided other criteria such as re-
agent, background, etc. are equal or similar in these two procedures.
The second situation is when a chemical confirmatory test yields a commonly known
derivative of known detector response. For example, in considering the confirmation
procedures such as kepone to mirex by chlorination, heptachlor to chlordene by
chromous chloride, and aldrin to dieldrin by expoxidation, the yield of the product
becomes significant since we know the detector response of these derivatives and a
higher yield of a derivative will give higher detector response.
Also, depending on the retention time, two peaks of equal peak areas can have
different peak heights. A broad peak will have a lower peak height than a sharper and
narrower peak of the same peak area. Obviously, at lower levels one can see a sharper
peak more easily than a broader one of the same area. In other words, the level for
detection or confirmation can be considerably lower for a sharper peak. For this rea-
son, peak height is mentioned as an important criteria rather than peak area.
In summary, disregarding the circumstance, for a confirmatory procedure by chem-
ical derivatization, comparison of the peak height of the product with that of the equiv-
alent concentration of the parent compound under the same GC conditions is more
indicative of its sensitivity and potential application than the yield of the reaction.
4. Simplicity, Applicability, and Specificity

Ideally a chemical confirmatory procedure should be simple to apply; thus many
tests can be handled almost at the same time by one person. It should be remembered
* Yield is a secondary consideration.

Volume 1 159
that for a procedure suitable to be used routinely, simplicity of procedure is an impor-

tant consideration. The procedure should be designed with more understanding of the
operational laboratory. It is not uncommon to see researchers lacking such understand-
ing to develop a method which is difficult to apply in practice. The problem is usually
not due to the chemical reaction but rather to the lack of understanding or sympathy
for the practical aspect. For example, a procedure may be fine for pure standard or
clean sample. At the higher level of concentration investigated or under the infrequent
or short periods of injections of reaction extracts during the investigation, the research-
ers may not observe any significant deleterious effect to the GLC system used to ex-
amine the reaction extracts. There may also be no investigation on or mention of the
possible interferences to the confirmation from the presence of other pesticides that
are possibly or commonly expected in the same sample. But, when this procedure is
applied to a routine laboratory, it may not only not work, but also it could affect the
GC column and detector to such an extent that the regular analysis is interrupted.
In addition to simplicity and applicability, a chemical confirmatory test should also
be specific to the compound in question. However, specificity of a procedure is not
necessarily limited to the concept that this procedure reacts only with the pesticide in
question and not with other pesticides that may be present. A procedure is considered
specific as long as derivatives from CO-extractivesand other pesticides show entirely
different analytical characteristics (e.g., GLC retention time or TLC R, values) from
the derivative in question.
VI. SUMMARY
The usefulness of chemical derivatization technique is not just confined to the con-
firmation of residue identity; in fact, this technique was first used for GC analysis of
less volatile or labile compounds that called for derivatization to more volatile or more
stable derivatives for successful G C analysis, particularly in the presence of sample co-
extractives. In fact, in the analysis of acid herbicides such as phenoxyalkanoic acids,
derivatization is necessary to permit GC analysis. For many N-methyl carbamates,
derivatization to more thermally stable compounds is required prior to GLC analysis.
There are many derivatization procedures for the analysis of acid herbicides and car-
bamates and they are discussed, respectively, in Volume 11, Chapter 3 , and Volume
111, Chapter 1 .
In conjunction with the increasing use of high pressure liquid chromatography
(HPLC) derivatization becomes a key step in applying this technique either before or
after H P L C column separation to compensate for the limitation in the choice of HPLC
detectors as compared to those available for GLC. As pointed out in a recent review,211
several recent review^^^^."^ and books327.328 on derivatization techniques for GLC or
HPLC analysis are available.
T o conclude, there are several approaches analysts can select to routinely confirm
identity of a compound in a sample. As pointed out in Volume 111, Chapter l the final
selection of a confirmatory approach and procedure or a combination of procedures
lies with the analyst and unfortunately personal preference and prejudice often plays
an important role in the selection. If money is available, there is a tendency among
analysts to persuade management to buy sophisticated and expensive equipment to do
a job whereas simpler and cheaper procedures or equipments can perform equally well.
In this regard, many analysts favor the GC/MS computer system rather than simpler
and much cheaper procedures such as chemical derivatization techniques, p-values,
and so on for confirming identity of pesticides in a routine laboratory even though
the need of such a system may be practically and operationally questionable for this
application. GC/MS computer system is no doubt a very versatile and useful tool. In
several types of activities such as research monitoring, it is the only effective tool to
d o the job. However, t o use it only for confirmation of identity in a routine operation
is overkill. Many laboratories in the world are using chemical and/or physical proce-
dures for confirmation of pesticides and other trace organic compounds in their rou-
tine operation, partly because of the capital and maintenance costs of a G U M S com-
puter system and partly because it is used more efficiently and effectively for those
areas, such as research, where such a system is really needed.
Before closing, it is no more than appropriate to quote a comment from my former
colleague who echos my sentiment when he remarkedzs5that: "In spite of all the in-
creases in sophisticated instrumentation connected with pesticide residue determina-
tions (such as the use of mass spectrometers) it is felt that a place still exists in pesticide
analysis for chemical reactions which can be used t o confirm pesticide residue assign-
ments. These chemical tests are simple and give positive identification quickly which
is important for routine operation." (The parenthesis was added by this author.)
REFERENCES
1. Reynolds, H. L., Use of gas chromatography by food and drug administration for pesticide residue
analysis, J . Gas Chromatogr., 2, 219, 1964.
2. Goulden, R., Goodwin, E. S., and Davis, L., Improvement of identification in the gas-liquid chro-
matographic analysis of agricultural samples for residues of some chlorinated pesticides, Analyst,
88, 941, 1963.
3. Robinson, J., Residues of organochlorine insecticides in dead birds in the United Kingdom, Chem.
Ind. (London), 1974, 1967.
4. Reynolds, L. M., PCB's and their interference with pesticide residue analysis, presented to the East-
ern Canada Seminar on Pesticide Residue Analysis, Guelph, Ontario, 1968.
5. Glotfeltz, D. E. and Caro, J. H., Artifacts of dieldrin in gas chromatographic analysis of green
material, J. Agric. Food Chem., 42, 284, 1970.
6. McLeod, H., Canadian Food and Drug Directorate, unpublished observation, 1967; Chau, A. S. Y.,
unpublished observation, 1968.
7. Pearson, J. R., Aldrich, F. D., and Stone, A. W., Identification of the aldrin artifact, J. Agric.
Food Chem., 15,938, 1967
8. Denbert, K. H., Sources of compounds interfering with the analysis of extracts for dieldrin and DDT
by GLC, Bull. Environ. Cont. Toxicol., 5, 379, 1970.
9. Mestres, R., Berthes, F., Priu, F., Residus de pesticides. Sur une cause derreur dans la recherche des
insecticides organochlores, Trav. Soc. Pharm. Montpellier, 26, 93, 1966.
10. Osadchuk, M. and Romach, M., Problems in interfering responses in the identification and quanti-
tation of pesticide residues, presented to the eastern Canada Seminar on Pesticide Residue Analysis,
Guelph, Ontario, 1968; McLeod, H., Ed., Analytical Methods for Pesticide Residue in Foods, Food
and Drug Directorate, Ottawa, Ontario, Canada.
11. Chau, A. S. Y., Derivative formation for the confirmation of endosulfan by gas chromatography,
J. Assoc. O f f .Anal. Chem., 52, 1240, 1969.
12. Chau, A. S. Y., Confirmation of pesticide residue identity. I. Derivative formation for the confir-
mation of photo products of endrin hexachloro and pentachloro-ketone pesticide residues by gas
chromatography, J. Assoc. O f f .Anal. Chem., 55,519, 1972.
13. Chau, A. S. Y. and Cochrane, W. P., Cyclodiene chemistry. I. Derivative formation for the identi-
fication of heptachlor, heptachlor epoxide, cis - chlordane, trans-chlordane, and aldrin pesticide res-
idues by gas chromatography, J. Assoc. O f f .Anal. Chem., 52, 1092, 1969.
14. Chau, A. S. Y. and Cochrane, W. P., Cyclodiene chemistry. 111. Derivative formation for the iden-
tification of heptachlor, heptachlor epoxide, cis - chlordane, trans-chlordane, dieldrin, and endrin
pesticide residues by gas chromatography, J. Assoc. O f f .Anal. Chem., 52, 1220, 1969.
15. Chau, A. S. Y., Some reactions of CrC12on organochlorine pesticides and their utilization in chemical
confirmative tests, presented to the Eastern Canada Seminar on Pesticide Residue Analysis, Ottawa,
Ontario, May, 1970.
16. Chau, A. S. Y., Approach to the analysis of some organochlorine pesticides in water - some prob-
lems and some solutions, in Report of the 6th Pesticide Residue Analysis Seminar (Western Canada),
Alberta Department of Agriculture, Edmonton, Alberta, 1971.
17. Chau, A. S. Y. and Cochrane, W. P., Chromous chloride reduction. VI. Derivative formation for
the simultaneous identification of heptachlor and endrin pesticide residues by gas chromatography,
3. Assoc. Off. Anal. Chem., 54, 1124, 1971.
18. Holdrinet, M., Ontario Ministry of Agriculture and Foods, Guelph, Ontario, private communication,
1971.
19. Beroza, M. and Bowman, M. C., ldentification of pesticides at nanogram level by extraction of p-
values, Anal. Chern., 37, 291, 1965.
20. Beroza, M. and Bowman, M. C., Extraction of insecticides for cleanup and identification, J. Assoc.
Anal. Chem.,48, 358, 1965.
21. Beroza, M. and Bowman, M. C., Apparatus and procedures for rapid extraction and identification
in binary solvent systems, Anal. Chem., 38, 837, 1966.
22. Beroza, M. and Bowman, M. C., Extraction p-values of pesticides and related compounds in six
binary solvent systems, 3. Assoc. Off. Anal. Chem.,48,943, 1965.
23. Beroza, M. and Bowman, M. C., Correlation of pesticide polarities with efficiencies of milk extrac-
tion procedures, 3. Assoc. Off. Anal. Chem.,49,1007,1966.
24. Beroza, M., Inscoe, M. N., and Bowman, M. C., Distribution of pesticides in immiscible binary
solvent systems for cleanup and identification and its application in the extraction of pesticides from
milk, Res. Rev., 30, 1, 1969.
25. Bowman, M. C. and Beroza, M., Device and method for determining extraction p-values with une-
quilibrated solvents or unequal phase volumes, Anal. Chem., 38, 1427, 1966.
26. Bowman, M. C. and Beroza, M., Identification of compounds by extraction p-values using gas chro-
matography, Anal. Chem.,38, 1544, 1966.
27. Elgar, K. E., ldentification of pesticides at residue concentrations - some problems and some solu-
tions, in The Identification of Pesticides at Residue Concentrations, Advances in Chemistry Series
104, American Chemical Society, Washington, D.C. 1971, 151.
28. Holden, A. V. H. and Marsden, K. M., The examination of surface waters and sewage effluents for
organo-chlorine pesticides, Proc. Inst. Sew. Purif. (London), p. 295, 1966.
29. McCully. K. A., Analysis of multiple pesticide residues - chemical identity and confirmation of
results, World Rev. Pest. Control, 8, 59, 1969.
30. Banks, K. A. and Bills, D. D., Gas chromatographic identification of chlorinated insecticides based
on their U.V. degradation, I. Chromatogr., 33,450, 1968.
31. Bills, D. D., Kaufman, W. M., and Hannan, E. J., Method for the identification of nanogram
amounts of certain insecticides, presented to the 162nd Nat. Meeting of Am. Chem. Soc., Washing-
ton, D.C., 1971.
32. Kaufman, W. M., Identification of Some Chlorinated Pesticides by Ultraviolet Degradation, Ph.D.
thesis, Oregon State University, University Microfilms, Ann Arbor, Michigan, June 1971.
33. Kaufman, W. M., Bills, D. D., and Hannan, Ed., Identification of nanogram amounts of certain
organochlorine insecticides, J. Agric. Food Chem., 20,628, 1972.
34. Beroza, M., Determination of the chemical structure of organic compounds at the microgram level
by gas chromatography, Ace. Chem. Res., 3,33, 1970.
35. Asai, R. I., Gunther, F. A., and Westlake, W. E., Carbon-skeleton chromatography for the qualti-
tative identification of selected insecticides, Res. Rev., 19, 57, 1967.
36. Saha, N. C. and Mathur, D. S., Studies on catalysts and catalysis by the techniques of gas chroma-
tography, 3. Chromatogr., 82,207, 1973.
37. Beroza, M., Determination of the chemical structure of microgram amounts of organic compounds
by gas chromatography, Anal. Chem., 34, 1801, 1962.
38. Beroza, M. and Sarmiento, R., Determination of the carbon-skeleton and other structural features
of organic compounds by gas chromatography, Anal. Chem., 35,1353, 1963.
39. Minyard, J. P. and Jackson, E. R., Gas chromatographic determination of flash heater-modified
pesticides, 3. Agric. Food Chem., 13.50, 1965.
40. Miller, G. A. and Wells, C. E., Alkaline pre-column for use in gas chromatographic pesticide residue
analysis, 3. Assoc. Off. Anal. Chem.,52, 548, 1969.
41. Chau, A. S. Y. and Wilkinson, R. J., unpublished investigation, 1972.
42. Luckas, B., Pscheidl, H. and Haberland, D., Determination of chlorinated pesticides and polychlor-
inated biphenyls by derivatization gas chromatography, 3. Chromatogr., 147,41, 1978.
43. Acklin, W. and Prelog, V., Reaktionen mit Mikroorganismen. IX. Die Bestimmung der absoluten
Konfiguration von 8-methyl-hydrindan-derivaten durch asymmetrische Synthese. Eine intramoleku-
lare 1,s-hydrid-verschiebung in der cis-hydrindan-reike, Helvetica Chim. Acta, 42, 1239, 1959.
44. Landa, S. and Markovec, L., Uber die Verschiebung dev Dopplebindungen in einigen Olefinen bei
der Chromatographie an Silicagel und Aluminumoxid, Coll. Czechoslov. Chem. Commun., 29, 2309,
1964.
45. Matsumoto, T., Fukuoka, Y., Ichihara, A., Mori, Y., Shirahama, H., Takahashi, Y., and Watanabe,
M., The stereochemistry of llludin S(Lampterol), Chem. Soc. Jpn. Bull., 37, 1716, 1964.
46. Barnes, R. A. and Lira, E. P., Rearrangement of a chloromethylketone on alumina, Chem. Ind.
(London), p. 2100, 1964.
47. Story, P. R. and Fahrenholtz, S. R., 7-Quadricyclo (2.2.1.0' 603.5]heptanone (Quadricyclanone), J.
Am. Chem. Soc., 86, 1270,1964.
48. Buchi, G. and Loewenthal, H. J. E., Synthesis of epicyclocolorenone and stereochemistry of cyclo-
colorenone, Proc. Chem. Soc., (London), p. 280, 1962.
49. Markovec, L. and Landa, S., Uber die Verschiebung der Doppelbindung in einigen Olefinen bei der
Chromatographie an Silicagel und Aluminiumoxid. 11. Stoukturivandel der Trialkylathylene und des
2,4,4 - trimethyl - l - pentens, Coll. Czechoslov. Chem. Commun., 30, 3672, 1965.
50. Herout, V., Banassek, A., and Romanuk, M., On terpenes. CLXXX. To the problem of identity of
P-cadinene, Coll. Czechoslov. Chem. Commun., 31,3012, 1966.
51. Benesova, V., Herout, V., and Sorm, F., On terpenes. CXXVIII. The existence of a- and p-humu-
lene, Coll. Czechoslov. Chem. Commun., 26,1832, 1960.
52. Muchowski, J. M., The use of alumina in the synthesis of a-diazo carbonyl compounds, Tetrahedron
Lett., 16, 1773, 1966.
53. Subba Rao, H. N., Damodaran, N. P., and Dev, S., Photochemistry of zerumbone, Tetrahedron
Lett., 3, 227, 1967.
54. Sobti, R. R. and Dev, S., Biogenetic-type transformation in diterpenoids, Tetrahedron Lett., 33,
3939, 1966.
55. Joshi, V. S., Damodaran, N. P., and Dev, S., Organic reactions in a solid matrix. I. Alumina-induced
oxirane rearrangements, Tetrahedron, 24,5817, 1968.
56. Boswell, G. A., Jr., Synthesis of 5a,6,6-trifluoro-3-keto and 6.6-difluoro-A'-3-keto steroids, J. Org.
Chem.,31,991, 1966.
57. Castells, J. and Fletcher, G. A., The hydrolysis of 3:s-dinitrobenzoates, J . Chem. Soc. (London), p.
3245, 1956.
58. Meinwald, J., Shelton, J. C., Buchanan, G. I., and Courtin, A., Highly strained bicyclic systems.
XII. Synthesis and solvolysis of 1.5.5-trimethylbicyclo {2.1,1.) hex-2-yl p-toluenesulfonate, J. Org.
Chem., 33,99, 1968.
59. Chau, A. S. Y., unpublished investigation, 1970.
60. Chau, A. S. Y. and Lanouette, M., Confirmation of pesticide residue identity. 11. Derivative forma-
tion in solid matrix for the confirmation of DDT, DDD methoxychlor, perthane, cis- and trans-
chlordane, heptachlor and heptachlor epoxide pesticide residues by gas chromatography, J. Assoc.
O f f .Anal. Chem.,55, 1058, 1972.
61. Chau, A. S. Y. and Terry, K., Confirmation of pesticide residue identity. IV. Derivative formation
in sold matrix for the confirmation of a- and p- endosulfan by gas chromatography, J. Assoc. Off.
Anal. Chem., 55, 1228, 1972.
62. Chau, A. S. Y., Confirmation of pesticide residue identity. V. Alternative procedure for derivative
formation in solid matrix for the confirmation of a- and /3-endosulfan by gas chromatography, J.
Assoc. O f f .Anal. Chem., 55, 1232, 1972.
63. Chau, A. S. Y. and Terry, K., Confirmation of pesticide residue identity. VI. Derivative formation
in solid matrix for confirmation of heptachlor and endosulfan isomers, J. Assoc. O f f .Anal. Chem.,
57, 395, 1974.
64. Chau, A. S. Y., Confirmation of pesticide residue identity. 111. Derivative formation in solid matrix
for the confirmation of endrin by gas chromatography, Bull. Environ. Cont. Toxicol., 8, 169, 1972.
65. Chau, A. S. Y., Confirmation of pesticide residue identity. VII. solid matrix derivation procedure
for the simultaneous confirmation of endrin and heptachlor residues in the presence of large quan-
tities of polychlorinated biphenyls, J . Assoc. O f f .Anal. Chem., 57,585, 1974.
66. Chau, A. S. Y. and Larose, R., unpublished results, 1971.
67. Stijve, T. and Cardinale, E., Confirmation of identity of heptachlor residues by hydroxy derivatiza-
tion on thin-layers of alumina oxide, Trav. Chim. Aliment. Hyg., 63, 308, 1972.
68. Burke, S . A., Ed., Pesticide Analytical Manual, Vol. I, Department of Health, Education and Wel-
fare, Food and Drug Administration, Washington, D.C., 1968.
69. Analytical Methods Manual, Inland Water Directorate, Water Quality Branch, Environment Canada,
Ottawa, Canada, 1974.
70. Horwitz, W., Ed., Official Methods of Analysis of the Association of Official Analytical Chemists,
Association of Official Analytical Chemists, Arlington, Virginia, 1975.
71. Kunze, F. M., Simplified procedure for the analysis of methoxychlor in fruits and vegetables, dairy
products, and biological materials, J. Assoc. O f f .Agric. Chem., 37, 578, 1954.
72. McKinley, W. P. and Graham, S. I., Identification of pesticide residues in extracts of fruit, vegetables
and animal fats. 11. Rapid quantitative chemical tests for captan and methoxychlor, J. Assoc. O f f .
Agric. Chern., 43, 89, 1960.
73. Prickett, C. S., Kunze, F. M., and Lang, E. P., Modification of the Schecter method for the deter-
mination of methoxychlor or DDT in biological materials, J. Assoc. O f f . Agric. Chern., 33, 880,
1950.
74. Miles, J. R. W., Chemical determination of perthane residues on agricultural crops, J. Agric. Food
Chern., 5,349, 1957.
75. Klein, A. K., Watts, J. O . , and Daminco, J. N., Chlorinated insecticides and miticides. Electron
capture gas chromatography for determination of DDT in butter and some vegetable oils, J. Assoc.
O f f .Chern., 46, 165, 1963.
76. Klein, A. K. and Watts, J. O., Separation and measurement of perthane, DDD(TDE) and DDT in
leafy vegetables by electron capture gas chromatography, J. Assoc. O f f .Agric. Chem., 47, 3 11, 1964.
77. Hammence, J. H., Hall, P. S., and Caverly, D. J., The identification and determination of chlori-
nated pesticide residues, Analyst, 90,649, 1965.
78. Sans, W. W., Multiple insecticide residue determination using column chromatography, chemical
conversion, and gas liquid chromatography, J. Agric. Food Chern., 15, 192, 1967.
79. Duffy, J. R. and Wong, N., Residues of organochlorine insecticides and their metabolites in soils in
the Atlantic Provinces of Canada, J. Agric. Food Chern., 15, 457, 1967.
80. Mendoza, C. E., Wales, P. J., McLeod, H. A., and McKinley, W. P., Sodium methylate treatment
of cleaned up plant extracts in confirmation of some pesticide residues by gas-liquid and thin-layer
chromatography, J. Assoc. O f f .Anal. Chern., 51, 1095, 1968.
81. Miller, G. A. and Wells, C. E., Alkaline pre-column for use in gas chromatographic pesticide residue
analysis, J. Assoc. O f f .Anal. Chern., 52, 548, 1969.
82. Pionke, H. B., Chesters, G., and Armstrong, D. E., Dual column and derivative techniques for
improved specificity of gas-liquid chromatographic identification of organochlorine insecticide resi-
dues in soils, Analyst, 94,900, 1969.
83. Marcos, G. B., Methodos para la extraccion de residuos de plaguicidas y metabolitos para la deter-
minacion multiple por cromatografia gas-liquido. B. Plaguicidas ionizables. C. Interferencias. D.
Purificacion de disolventes, Rev. Agroq. Tecnol. Alioyentos, 9, 536, 1969.
84. Chau, A. S. Y., Wilkinson, R. J., and Lanouette, M., unpublished results, 1971.
85. Krause, R. T., Quantitative dehydrochlorination of perthane residues and effect of alcoholic potas-
sium hydroxide on other pesticides, J. Assoc. O f f .Anal. Chern., 55, 1042, 1972.
86. Young, S. J. V. and Burke, J. A., Micro scale alkali treatment for use in pesticide residue confirma-
tion and sample cleanup, Bull. Environ. Cont. Toxicol., 7, 160, 1972.
87. Miles, J. R. W., Conversion of DDT and its metabolites to dichlorobenzophenones for analysis in
the presence of polychlorinated biphenyls, J. Assoc. O f f .Anal. Chern., 55, 1039, 1972.
88. Reynolds, L. M., Polychlorobiphenyls (PCB's) and their interference with pesticide residue analysis,
Bull. Environ. Cont. Toxicol., 4, 128, 1969.
89. Sims, G. G., Campbell, J. R., Zemlyak, F., and Graham, J. M., Organochlorine residues in fish
and fishery products from the Northwest Atlantic, Bull. Environ. Cont. Toxicol., 18, 697, 1977.
90. Chau, A. S. Y. and Cochrane, W. P., Chromous chloride reductions. 11. The facile degradation of
p,p'-DDT via p,p'DDD using chromous chloride as reducing agent, Bull. Environ. Cont. Toxicol.,
5, 133, 1970.
91. Dennis, W. H., Jr. and Cooper, W. J., Catalytic dechlorination of organochlorine compounds. I.
DDT, Bull. Environ. Cont. Toxicol., 14, 738, 1975.
92. Dennis, W. H., Jr. and Cooper, W. J.. Nickel boride catalyzed dechlorination of several organochlo-
rine pesticides, Technical Report 7702, U.S. Army Medical Research and Development Command,
Washington, D.C., April 1977.
93. Smith, J. G. and Talvitie, R., Confirmatory test for the identification of pesticide residues, Project
No. 3016-2, Office of Research Administration, University of Waterloo, Research Institute, Water-
loo, Ontario, 1976.
94. Lewis, G. N., Magel, T. T., and Lipkin, D., The absorption and re-emission of light by cis- and
trans-stilbenes and the efficiency of their photo-chemical isomerization, J. Am. Chern. Soc., 62,
2973, 1940.
95. DeTar, D. F. and Carpino, L. A., The preparation of and the spectral characteristics of some 2-
substituted cis- and trans-stilbenes, J. Am. Chem. Soc., 78,475, 1956.
96. Glotfelty, D. E., Identification of organochlorine pesticide residues by ultraviolet solid-phase pho-
tolysis, Anal. Chern.,44, 1250, 1972.
97. Lindquist. A. W., Jones, H. A.. and Madden, A. H., DDT residual-type sprays as affected by light,
J. Econ. Entomol., 39, 55, 1946.
98. Wichmann, H. J., Patterson, W. I., Clifford, P. A., Klein, A. K., and Claborn, H. V., Decomposi-
tion and volatility of DDT and some of its derivatives, J. Assoc. O f f .Agric. Chern., 29, 218, 1946.
99. Kennedy, M. V., Stojanovic, B. J., and Shuman, F. L., Jr., Analysis of decomposition products of
pesticides, J . Agric. Food Chem., 20, 341, 1972.
100. Fleck, E. E., The action of ultraviolet light on DDT, J. Am. Chem. Soc., 71, 1034, 1949.
101. Grummitt, O., Buck. A., and Steams, J., 1,1-Di-pchlorophenyl)-1,2,2,2,-tetra-chloroethane, J. Am.
Chem. Soc., 67, 155, 1945.
102. Haller, H. L., Bartlett, P. D., Drake, N. L., Newman, M. S., Cristol, S. J., Eaker, C. M., Hayes,
R. A., Kilmer, G. W., Magerlein, K. B., Mueller, G. P., Schneider A., and Wheatly, W., The chem-
ical composition of technical DDT, J . Am. Chem. Soc., 67, 1591, 1945.
103. Cristol, S. J., Soloway. S. B., and Haller, H. L., Isolation of o,o'-DDT isomer from technical DDT,
J. Am. Chem. Soc., 69,5 10, 1947.
104. Forrest, J., Stephenson, O., and Waters, W. A., Chemical investigation of insecticide "DDT" and
its analogues. I. Reactions of "DDT" and associated compounds, J . Chem. Soc. (London), 333,
1946.
105. Gunther, F. and Blinn, R. C., Ultraviolet spectrophotometric microdetermination of acaricide 4.4'-
dichloro-a-(trichloromethyl) benzhydrol (FW-293), J . Agric. Food Chem., 5, 517, 1957.
106. Phillips, D. D., Pollard, G. E., and Soloway, S. B., Thermal isomerization of endrin and its behav-
iour in gas chromotography, J . Agric. Food Chem., 1,217, 1962.
107. Rosen, J. D., Sutherland, D. J., and Lipton, G. R., The photochemical isomerization of dieldrin
and endrin and effects on toxicity, Bull. Environ. Cont. Toxicol., 1, 133, 1966.
108. Zabik, M. J., Schuetz, R. D., Burton, W. L., and Pape, B. E., Photochemistry of bioactive com-
pounds. Studies of a major photo lytic product of endrin, J . Agric. Food Chem., 19, 308, 1971.
109. Roburn, J., Effect of sunlight and ultraviolet radiation on chlorinated residues, Chem. Ind. (Lon-
don), p. 1555, 1963.
110. Bird, C. W., Cookson, R. C., and Crundwell, E., Cyclisations and rearrangements in the isodrin-
aldrin series, J . Chem. Soc. (London), p. 4809, 1961.
111. Woodham, D. W., ldentification of the gas chromatographic endrin peak by its conversion to delta-
keta endrin, Report No. ARS 81-16, U.S. Department of Agriculture, Hyattsville, Maryland, 1967.
112. Woodham, D. W., Loftis, C. D., and Collier, C. W., Identification of the gas cromatographic diel-
drin and endrin peaks by chemical conversion, J. Agric. Food Chem., 20, 163, 1972.
113. Soloway, S. B., Damiana, A. M., Sims, J. W., Bluestone, H., and Lidov, R. E., Skeletal rearrange-
ments in reactions of isodrin and endrin, J . Am. Chem. Soc., 82, 5377, 1960.
114. Skerrett, E. J. and Baker, E. A., The microdetermination of endrin, Analyst, 85,606, 1960.
115. Fahey, J. E. and Schechter, M. S., Diazotized sulfannilic acid reagent for endrin analysis, J. Agric.
FoodChem.,9,192,1961.
116. Chau, A. S. Y., Confirmation of pesticide residue identity, presented to the 85th Annu. Meet. Assoc.
Off. Chem., October 11 to 14, Association of Official Chemists, Washington, D.C., 1971.
117. Chau, A. S. Y. and Wilkinson, R. J., Some separation characteristics of an OV-101/OV-210 column
for organochlorinated pesticides with particular reference to the separation of photoendtin and en-
drin, Bull. Environ. Cont. Toxicol., 7,93, 1972.
118. Chau, A. S. Y., Solid matrix derivatization technique. I. Derivative formation in solid matrix for
the confirmation of endrin by gas chromatography, in Proc. 7th Pesticide Residue Analysis Seminar
(Western Canada), Department of Agriculture, Saskatoon, 1972.
119. Wiencke, W. W. and Burke, J. A., Derivatization of dieldrin and endrin for confirmation of residue
identity, J. Assoc. Off. A n a l Chem.,52, 1277, 1969.
120. Chau, A. S. Y., Chromous chloride reductions. IV. Reaction of endrin with chromous chloride so-
lution, Bull. Environ. Cont. Toxicol., 5, 435, 1970.
121. Cochrane, W. P. and Chau, A. S. Y., Chemical derivatization techniques for confirmation of orga-
nochlorine residue identity, in Pesticides Identification at Residue Level, Gould, R. F., Ed., Advances
in Chemistry Series, No. 104, American Chemical Society, Washington, D.C., 1971, chap. 2.
122. Chau, A. S. Y., Won, H., and Cochrane, W. P., Chromous chloride reductions. V. Reaction of
endrin ketone (11) with chromous chloride solution, Bull. Environ. Cont. Toxicol., 6,481, 1971.
123. Chau, A. S. Y. and Wilkinson, R. J., Chromous chloride reduction. VII. Stereoc hemistry and stru-
cutre of the major product employed in the confirmation of endrin residues, Bull. Environ. Cont.
Toxicol., 8, 105, 1972.
124. Chau, A. S. Y. and Cochrane, W. P., Identification of derivatives employed in the confirmation of
dieldrin residues, Bull. Environ. Cont. Toxicol., 6, 515, 1971.
125. ApSimon, J. W., Buccini, J . A., and Chau, A. S. Y., The acid-catalyzed Wagner-Meerwein rear-
rangement of dieldrin, Tetrahedron Lett., p. 539, 1974.
126. Musial, C. J., Peach, M. E., and Stiles, D. A., A simple procedure for the confirmation of residues
of a- and p-endosulfan, dieldrin, endrin and heptachlor epoxide, Bull. Environ. Cont. Toxicol., 16,
98, 1976.
127. Maybury, R. B. and Cochrane, W. P., Evaluation of chemical confirmatory tests for analysis of
dieldrin residues, J . Assoc. Off. Anal. Chem., 56, 36, 1973.
Volume I 165
128. O'Donnell, Johnson, A. E., Jr., and Weiss, F. T., Chemical determination of dieldrin in crop mate-
rials, J. Agric. Food Chem., 3,151, 1955.
129. Skerrett, E. J. and Baker, E. A., The colorimetric determination of dieldrin in extracts of coffee
bark, Analyst, 84, 376, 1959.
130. Gunther, F. A., Kolbezen, M. J., and Blinn, R. C., A preliminary report on the colorimetric detection
of dieldrin, J. Econ. Entomol.,41, 185, 1954.
131. Parson, A. M. and Koore, D. J., Some reactions of dieldrin and the proton magnetic resonance
spectra of the products, /. Chem. Soc. (London), (c), 2026, 1966.
132. Elgar, K. E., Identification of pesticides at residue concentrations — some problems and some solu-
tions in Pesticides Identification at Residue Level, Advances in Chemistry Series, No. 104, Gould,
R. F., Ed., American Chemical Society, Washington, D.C., 1971.
133. Noren, K., Determination of aldrin residues in vegetables by the chemical conversion of aldrin to
dieldrin, Analyst(London), 93, 39, 1968.
134. Osadchuk, M. and Wanless, E. B., Identification and quantitative estimation of aldrin residues in
the presence of interfering materials in samples of plant and animal origin, /. Assoc. Off. Anal.
Chem. ,51, 1264, 1968.
135. Cochrane, W. P. and Chau, A. S. Y., unpublished results, 1969.
136. Chau, A. S. Y. and Wilkinson, R. J., unpublished results, 1970.
137. Chau, A. S. Y. and Tze, H., unpublished results, 1973.
138. Soloway, S., U.S. Patent 2,676,131, 1954; Chem. Abstr.,48, 8473, 1954.
139. Davies, D. I., The addition of bromotrichtoromethane and carbon tetrachlorine to aldrin, J. Chem.
Soc. (London),?. 3669, 1960.
140. Mackenzie, K., Bicyclo {2,2,1} heptadiene in the Diels-Alder Reaction, J. Chem. Soc. (London), p.
473, 1960.
141. Chau, A. S. Y. and Babjak, L., Column chromatographic determination of mirex, photomirex and
polychlorinated biphenyts in lake sediments, J. Assoc. Off. Anal. Chem.,62, 107, 1979.
142. Riemschneider, R., The Chemistry of the insecticides of the diene group, World Rev. Pest Cont., 5,
29, 1966.
143. Soloway, S. B., Correlation between biological activity and molecular structure of the cyclodiene
insecticides, in Advances in Pest Control Research, Metcalf, R. L., Ed., Interscience, New York,
1965,85.
144. Fleck, E. E., Chemistry of insecticides, in Pesticides and Their Effects on Soils and Water, Special
Pub. No. 8, Soil Science Society of America, Champaign, 111., 1966.
145. Metcalf, R. L., Organic Insecticides—Their Chemistry and Mode of Action, Interscience, New York,
1955, Chap. 10.
146. Ungnade, H. E. and McBee, E. T., The chemistry of perchlorocyclopentenes and cyclopentadienes,
Chem. Kev.,58, 249, 1958.
147. Melnikou, N. N., Chemistry of pesticides, Res. Rev.,36, 52, 1971.
148. Ward, P. M., Confirming heptachlor and heptachlor epoxide in food samples by gas-liquid chroma-
toegraphy of their photoderivatives, J. Assoc. Off. Anal. Chem.,60, 673, 1977.
149. Cochrane, W. P. and Chau, A. S. Y., Note on gas chromatographic indentification of heptachlor
pesticide residues by derivative formation. J. Assoc. Off. Anal. Chem.,51, 1267, 1968.
150. Cochrane, W. P. and Chau, A. S. Y., Use of chromous chloride for the confirmation of heptachlor
residues by derivatization, Bull. Environ. Cont. Toxicol.,5, 251, 1970.
151. Chau, A. S. Y., Chromous chloride reductions. III. Identification of products obtained from pro-
longed contact of chlordene and heptachlor with chromous chloride solution, Bull. Environ. Cont.
Toxicol.,5,435, 1970.
152. Vogelbach, C., Isolierung neuer kristalliner Substanzen aus technischen Chlordans, Angew. Chem.,
63,378, 1951.
153. March, R. B., Resolution and chemical and biological characterization of some constituents of tech-
nical chlordane, J. Econ. Entomol.,45, 452, 1952.
154. Shogam, S. M., Efimenko, I. A., Nikiforova, N. M., and Mel'nichenko, E. L., The chromatographic
analysis of industrial heptachlor, Zh. Anal. Khim., 17, 260, 1962.
155. Polen, P. B., Chlordane-composition analytical considerations and terminal residues, submitted to
IUPAC commission on Terminal Residues, Geneva, 1966.
156. Hurtig, H., Working paper on chlordane, prepared for FAO/WHO Joint Meeting on Pesticide Res-
idues, November 29 to December 11, Department of Agriculture, Ottawa, 1967.
157. Benvenue, A. and Yeo, C. Y., Gas chromatographic characteristics of chlordanes. I. Effect of an
aqueous invironment on the heptachlor component, Bull. Environ. Cont. Toxicol., 4, 68, 1969; Ben-
venue, A. and Yoe, C. Y., Gas chromatographic characteristics of chlordane. II. Observed compo-
sitional changes of the pesticide in aqueous and non-aqueous environments, J. Chromatogr., 42, 45,
1969.
158. Saha, J. G. and Lee, Y. W., Isolation and identification of the components of a commercial chlor-
dane formulation, Bull. Environ. Cont. Toxicol.,4, 285, 1969.
159. Polen, P. B., Terminal chlordane residues - evaluation, in IUPAC commission on terminal residues,
K. B. Hill (secretary to commission), J. Assoc. Off. Anal. Chem., 54, 1318, 1971.
160. Velsicol Chemical Corp., Standard for Technical Chlordane, Technical Bulletin, Velsicol Chemical
Corp., Chicago, Illinois, August 1971.
161. Subcommittee on pesticides and related compounds, Chlordane - its effects on Canadian ecosystems
and its chemistry, Report no. 2, NRCC No. 14094, NRC Associate Committee on Scientific Criteria
for Environmental Quality, National Research Council of Canada, Ottawa, (no date).
162. Mills, P. A., Bong, B. A., Kamps, L. R., and Burke, J. A., Elution solvent system for Florisil column
clean-up in organochlorine pesticide residue analyses, J. Assoc. Off. Anal. Chem., 55, 39, 1972.
163. Berg, 0. W., Diosady, P. E., and Rees, G. A. V., Column chromatographic separation of polychlor-
inated biphenyls from chlorinated hydrocarbon pesticides, and their subsequent gas chromatographic
quantitation in terms of derivatives, Bull. Environ. Cont. Toxicol., 7, 338, 1972.
164. Singh, J., Conversion of heptachlor to its epoxide, Bull. Environ. Cont. Toxicol.,4,77, 1969.
165. Chau, A. S. Y., unpublished result, 1976.
166. Armour, J. A. and Burke, J. A., Method for separating polychlorinated biphenyls from DDT and
its analogs, J. Assoc, Off. Anal. Chem., 53,761, 1970.
167. Cochrane, W. P., Forbes, M., and Chau, A. S. Y., Cyclodiene chemistry. Assignment of configura-
tion of two nonachlors via synthesis and derivatization, J. Assoc. Off. Anal. Chem., 53, 769, 1970.
168. Greve, P. A. and Wit, S. L., Rapid identification method for endosulfan from Glc peak shifts under
the influence of alkali, J . Agric. Food Chem., 19, 372, 1971.
169. Maier-Bode, H., Nacherntefaktoren und die Ruckstande, Res. Rev., 22, 1, 1968.
170. Gorbach, S. G., Christ, 0.E., and Kellner, H. M., Metabolism of endosulfan in milk sheep, J .
Agric. Food Chem., 19,950, 1968.
171. Schuphan, I., Ballshmiter, K., and Tolg, G., Zurn Metabolismus des Endosulfans in Ratten and
Mausen, Z. Naturforsch., 23b, 701, 1968.
172. Archer, T. E., Nazer, I. K., and Crosby, D. G., Photodecomposition of endosulfan and related
products in thin films by ultraviolet light irradiation, J . Agric. Food Chem., 20, 954, 1972.
173. Barnes, W. W. and Ware, G. W., The absorption and metabolism of C"- labeled endosulfan in the
house fly, J. Econ. Entomol., 58, 286, 1965.
174. Gorbach, S. G., Christ, 0. E., Kellner, H. M., Kloss, G., and Borner, E., Metabolism of endosulfan
in milk sheep, J . Agric. Food Chem., 16,950, 1968.
175. Ruckert, W. and Ballschmiter, K., Metabolismus der cyclodien-insektizide Alodan and Endosulfan
(-Thiodan) in Fliegen, Z. Anal. Chem., 259, 188, 1972.
176. Forman, S. E. and Graham, J. R., Autoradiolysis of 6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexa-
hydro-6.9,-methano-2,4,3-benzodioxathiepine 3-oxide-5a,9a-C", J. Org. Chem., 29, 233, 1964.
177. Forman, S. E., Durbetaki, A. J., Cohen, M. V., and Olofson, R. A., Conformational equilibria in
cyclic sulfites and sulfates. The configurations and conformations of the two isomeric Thiodans, J.
Org. Chem., 30, 169, 1965.
178. Martens, R., Der abbau von Endosulfan durch Mikroorganism des Bodens, Schriftenr. Ver. Wasser-
Boden-Lufthyg., Berlin-Dahlem, 37, 167, 1972.
179. Ballschmiter, K. and Tolg, G., Metabolism of Thiodan in insects, Angew Chem. Int. Ed. Eng., 5,
730, 1966.
180. Putnam, T. B., Bills, D. D., and Libbey, L. M., Identification of endosulfan based on the products
of laboratory photolysis, Bull. Environ. Cont. Toxicol., 13,662, 1975.
181. Task Force on Mirex, Mirex in Canada, Technical Report 77-1, Appendix 1, Fisheries and Environ-
ment Canada, Health and Welfare Canada, 1977, 107.
182. Chau, A. S. Y., Carron, J. M., and Tse, H., Confirmation of pesticide residues identity. X. Mirex,
J. Assoc. Off. Anal. Chem.,61, 1475, 1978.
183. Berg, 0. W., P. L. Diosady, and Rees, G. A. V., Column chromatographic separation of polychlor-
inated biphenyls from chlorinated hydrocarbon pesticides, and the subsequent gas chromatographic
quantitation in terms of derivatives, Bull. Environ. Cont. Toxicol., 7, 338, 1972.
184. Huntzinger, Q., Safe, S., and Zitko, V., Analysis of Chlorinated aromatic hydrocarbons by exhaus-
tive chlorination - quantitative and structural aspects of the perchloro-derivatives of biphenyl, nap-
thalene, terphenyl, dibenzofuran, dibenzodioxin and DDE, Int. J. Environ. Anal. Chem., 2, 95,
1972.
185. Huckins, J. N., Swanson, J. E., and Stalling, D. L., Perchlorination of polychlorinated biphenyls,
J . Assoc. Off. Anal. Chem.,57,416, 1974.
186. Trotter, W. J, and Young, S. J. V., Limitation on the use of antimony pentachloride for perchlori-
nation of polychlorinated biphenyls, J. Assoc. Off. Anal. Chem., 58, 466, 1975.
187. Amour, J. A., Quantitative perchlorination of polychlorinated biphenyls as a method for confirma-
tion residue measurement and identification, J. Assoc. Off. Anal. Chem., 56,987, 1973.
188. Stratton, C. L., Allan, J. M., and Whitlock, S. A., Advances in the application of the perchlorination
technique for the quantitation and confirmation of polychlorinated biphenyls (PCBs), Bull. Environ.
Cont. Toxicol., 21, 230, 1979.
189. Widmark, G., Possible interference by chlorinated biphenyls, in IUPAC commission on methods of
pesticide residue analysis, J. Assoc. Off. Anal. Chern., 50, 1069, 1967.
190. Jensen, S. and Widmark, G., Organochlorine Residues, OECD preliminary study 1966-67, unpub-
lished report, 1967.
191. Risebrough, R. W., Reiche, P., and Olcott, H. S., Current progress in the determination of the
polychlorinated biphenyls, Bull. Environ. Cont. Toxicol., 4, 192, 1969.
192. Hallett, D. J., Norstrom, R. J., Onuska, F. I., Comba, M. E., and Sampson, R., Mass spectral
confirmation and analysis by Hall detector of mirex and photomirex in herring gulls from Lake
Ontario, J . Agric. Food Chern., 24, 1189, 1976.
193. Norstrom, R. J., Won, H. T., Holdrinet, M. V. H., Calway, P. G., and Naftel, C. D., J. Assoc.
Off. Anal. Chern., in press, 1979.
194. Lusby, W. R. and Hill, K. R., Detection of mirex in the presence of polychlorinated biphenyls, Bull.
Environ. Cont. Toxicol., 22,567, 1979.
195. Chau, A. S. Y., Analysis of mirex in lake sediment: problems and solution, presented to the Int.
Symp. on the Analysis of Hydrocarbons and Halogenated Hydrocarbons, McMaster University,
Hamilton, Ontario, May 23 to 25, 1978.
196. Chau, A. S. Y. and Thomson, R., Analytical reference materials. I. Preparation and purification of
photomirex (1,2,3,4,5,5,6,7, 10-undecachloropentacyclo [5.3.0.01.6.03.9.0'.8]decane, J. Assoc. Off.
Anal. Chem., in press, 1979.
197. Kecher, R. M., Skibinskaya, M. B., Gallai, 0. S. and Zefiros, N.S.. A new method of reducing
polyhalogenated compounds, (trans.), Zh. Org. Khirn., 10.41 1, 1974.
198. Kray, W. C., Jr. and Castro, C. E., The cleavage of bonds by low-valent transition metal ions. The
homogeneous dehalogenation of vicinal dihalides by chromous sulfate, J. Org. Chem., 86, 4603,
1964.
199. Singleton, D. M. and Kochi, J. K., The mechanism of reductive elimination of vic-dihalides by chro-
mium (II), J. Am. Chern. Soc., 89, 6547, 1967.
200. Kochi, J. K., Singleton, D. M., and Andrews, L. J., Alkenes from halides and epoxides by reductive
eliminations with Cr" complexes, Tetrahedron, 24, 3503, 1968.
201. Kochi, J. K. and Mocadlo, P. E., Facile reduction of alkyl halides with chromium (11) complexes, J.
Am. Chern. Soc., 88,4094, 1966.
202. Erickson, R. E. and Holmquist, Stereochemistry of Cr (11) reductions, Tetrahedron Lett., p. 4209,
1969.
203. Chau, A. S. Y., Demayo A., ApSimon, J. W., Buccini, J. A., and Fouchier, A., Chromous chloride
reduction. VIII. Reaction of some derivatives and degradation products of heptachlor with chromous
chloride-ethylenediamine complex and nuclear magnetic and mass spectra of the products, J. Assoc.
Off. Anal. Chern., 57,205, 1974.
204. Webb, R. G. and McCall, A. C., Identities of polychlorinated biphenyl isomers in Aroclors, J. Assoc.
Anal. Chern., 55, 746, 1972.
205. Alley, E. G., Dollar, D. A., Layton, B. R., and Minyard, J. P., Jr., Photochemistry of mirex, J .
Agric. FoodChern.,21, 138, 1973.
206. Alley, E. G., Layton, B. R., and Minyard, J. P., Jr., Photoreduction of mirex in aliphatic amines,
J. Agric. Food Chern., 22,727, 1974.
207. Alley, E. G., Layton, B. R., and Minyard, J. P., Jr., Identification of photoproducts of the insecti-
cides mirex and kepone, J. Agric. Food Chern., 22,442, 1974.
208. Lewis, R. G., Hamisch, R. C., MacLeod, K. E., and Sovocool, G. W., Photochemical confirmation
of mirex in the presence of polychlorinated biphenyls, J. Agric. Food Chern., 24, 1030, 1976.
209. Mes, J., Davies, D., and Miles, W., Traces of mirex in some Canadian human milk samples, Bull.
Environ. Cont. Toxicol., 19, 564, 1978.
210. Lane, R. H., Grodner, R. M., and Graves, J. L., Irradiation studies of Mallard duck eggs material
containing mirex, J. Agric. Food Chern., 24, 192, 1972.
21 1. Cochrane, W. P., Application of chemical derivatization techniques for pesticide analysis, J. Chro-
rnatogr. Sci., 17, 124, 1979.
212. Moseman, R. F., Ward, M. K., Crist, H. L., and Zehr, R. D., A micro derivatization technique for
the confirmation of trace quantities of kepone, J. Agric. Food Chern., 20,965, 1978.
213. Roberts, C. W., Chemistry of hexachlorocyclopentadiene, Chern. lnd., p. 110, 1958.
214. Spencer, E. Y., Guide to the Chemicals Used in Crop Protection, 6th ed., Research Branch, Agricul-
ture Canada, Ottawa, 1973.
215. Stijve, T., The determination and occurrence of hexachlorobenzene residues, Trav. Chim. Aliment.
Hyg., 62,406, 1971.
216. Cochrane, W. P. and Maybury, R. B., Chemical confirmation of BHC isomers: comparison of al-
kaline reactions in solution and by gas chromatographic pre-column, J. Assoc. O f f .Anal. Chem.,
56, 1324, 1973.
217. Davidow, B. and Woodard, G., An ultraviolet spectrophotometric method for the determination of
benzene hexachloride, J. Assoc. O f f .Agric. Chem., 32, 751, 1949.
218. Van der Linden, J. V., Uber die benzol-hexachloride und ihren zerfall in trichlor-benzole, Chem.
Ber.,45, 231, 1912.
219. Bradbury, F. R. and Standen, H., The calorimetric determination of benzene hexachloride in insect
tissues, J . Sci. Food Agric., 5, 252, 1954.
220. Levi, I., Mazur, P. B., and Nowicki, T. W., Rapid screening method for the determination of orga-
nochlorine pesticide residues in wheat by electron capture gas chromatography, J . Assoc. O f f .Anal.
Chem., 55,794, 1972.
221. McLeod, H. A. and Wales, P. J., A low temperature cleanup procedure for pesticides and their
metabolites in biological samples, J . Agric. Food Chem., 20, 624, 1972.
222. Dennis, W. H., Jr. and Cooper, W. J., Catalytic dechlorination of organochlorine compounds. 111.
Lindane, Bull. Environ. Cont. Toxicol., 18,57, 1977.
223. Larose, R. H., High-speed liquid chromatographic cleanup of environmental samples prior to GLC
determination of lindane, 3. Assoc. O f f .Anal. Chem., 57, 1046, 1974.
224. Stijve, T. and Cardinale, E., On the residue associated with the use of technical grade BHC with
special reference to the occurrence and determination of the three pentachlorocyclohex-I-ene isomers,
Trav. Chim. Aliment. Hyg.,63, 142, 1972.
225. Holdrinet, M., Determination and confirmation of hexachlorobenzene in fatty samples in the pres-
ence of other residual halogenated hydrocarbon pesticides and polychlorinated biphenyls, J. Assoc.
O f f .Anal. Chem.,57, 580, 1974.
226. Gehring, P. J. and MacDougall, D., Review of the Toxicity of Hexachlorobenzene and Hexachloro-
butadiene, Chemical Biology Research Toxicology Section, Dow Chemical, Midland, Mich.
227. Gilbertson, M. and Reynolds, L. M., Hexachlorobenzene (HCB) in the eggs of common terns in
Hamilton Harbour, Ontario, Bull. Environ. Cont. Toxicol., 7, 371, 1972.
228. Taylor, I. S. and Keenan, F. P., Studies on the analysis of hexachlotobenzene residues in food stuffs,
J . Assoc. O f f .Anal. Chem.,53, 1293, 1970.
229. Collins, G. B., Holmes, D. C., and Wallen, M., Identification of liexachlorobenzene residues by gas-
liquid chromatography, J. Chromatogr., 60, 198, 1972.
230. Crist, H. L., Moseman, R. F., and Noneman, J. W., Rapid determination and confirmation of low
levels of hexachlorobenzene in adipose tissue, Bull. Environ. Cont. Toxicol., 14, 273, 1975.
231. Brevik, E. M., Gas chromatographic method for the determination of organochlorine pesticides in
human milk, Bull. Environ. Cont. Toxicol., 19, 281, 1978.
232. Baker, B. E., Confirmation of hexachlorobenzene by chemical reaction, BUN. Environ. Cont. Toxi-
col., 10, 279, 1973.
233. Lee, B. and Chau, A. S. Y., unpublished observation, 1979.
234. Chau, A. S. Y. and Coburn, J. A., Determination of pentachlorophenol in natural and waste waters,
3. Assoc. O f f .Anal. Chem., 57,389, 1973.
235. St. John, L. E., Jr. and Lisk, D. J., Determination of hydrolytic metabolites of organophosphorus
insecticides in cow urine using an improved thermionic detector, 3. Agric. Food Chem., 16, 48, 1968.
236. Askew, J., Ruzicka, J. H., and Wheals, B. B., Organophosphorous pesticides - a gas chromato-
graphic screening technique based on detection of methylated hydrolysis products, J. Chromatogr.,
41, 180, 1969.
237. Shafik, M. T., Bradway, D., and Enos, E. F., A method for confirmation of organophosphorus
compounds at the residue level, Bull. Environ. Cont. Toxicol., 6,55, 1971.
238. Nesterova, I. P., Use of methylation reaction for gas chromatographic determination of organophos-
phorus pesticides, Zh. Anal. Khim., 32, 1790, 1977.
239. Cochrane, W. P., Greenhalgh, R., and Looney, N. E., Gas-liquid chromatographic analysis of ethe-
phon and fenoprop residues in apples and their decline before and after harvest, 3. Assoc. O f f .Anal.
Chem., 59,617, 1976.
240. Dale, W. E., Miles, J . W., and Churchill, F. C., 11, Gas-liquid chromatographic determination of
chlorophoxim residues in water and fish by in-block methylation, J. Assoc. O f f .Anal. Chem., 59,
1088, 1976.
241. Churchill, F. C., 11, Ku, D. N., and Miles, J. W., Gas-liquid chromatographic inlet block derivatis-
ation of organophosphorus pesticides and related dialkyl phosphorothioates, J. Agric. Food Chem.,
26, 1108, 1978.
242. Miles, J . W. and Dale, W. E., Gas chromatographic determination of organophosphorus pesticides
by in-block methylation, J . Agric. Food Chem., 26,480, 1978.
243. Moye, H. A., Reaction gas chromatographic analysis of pesticides. I. On-column transesterification
of n-methylcarbamates by methanol, J . Agric. Food Chem., 19,452, 1971.
Volume I 169
244. Moye, H. A., Reaction gas chromatographic analysis of pesticides. 11. On-column transesterification
of organophosphates by methanol, J. Agric. Food Chem., 21,621, 1973.
245. Tanaka, F. S. and Wien, R. G., Gas chromatography of substituted phenylureas by flash-heater
methylation with trimethylaniluim hydroxide, J . Chromatogr., 87, 85, 1973.
246. Bromilow, R. H. and Lord, K. A., Analysis of sulfur-containing carbamated by formation of deriv-
atives in the gas-liquid chromatograph using trimethyl-ammonium hydroxide, J. Chromatogr., 125,
495, 1976.
247. Kawahara, F. K., Micordetermination of derivatives of phenols and mercaptans by means of electron
capture gas chromatography, Anal. Chem., 40, 1009, 1968.
248. Kawahara, F. K., Microdetermination of pentafluorobenzyl ester derivatives of organic acids by
means of electron capture gas chromatography, Anal. Chem., 40, 2073, 1968.
249. Kawahara, F. K., Gas chromatographic analysis of mercaptans, phenols, and organic acids in surface
water with use of pentafluorbenzyl derivatives, Environ. Sci. Technol., 5, 235, 1971.
250. Kawahara, F. K., Trace organic components as fingerprints in gas chromatographic identification
of spilled asphalts, Environ. Sci. Technol., 10, 761, 1976.
251. Johnson, L. G., Formation of pentafluorobenzyl derivatives for the identification and quantitation
of acid and phenol pesticide residues, J . Assoc. O f f .Anal. Chem., 56, 1503, 1973.
252. McCallum, N. K. and Armstrong, R. J., The derivatization of phenols for gas chromatography using
electron capture detection, J. Chromatogr., 78, 303, 1973.
253. Sieber, J . N., Crosby, D. G., Fonda, H., and Soderquist, C. J., Ether derivatives for the determina-
tion of phenols and phenol-generating pesticides by electron capture gas chromatography, J . Chro-
matogr., 73, 89, 1972.
254. Coburn, J. A. and Chau, A. S. Y., Confirmation of pesticide residue identity. V111. Organosphorus
pesticides, J . Assoc. O f f .Anal. Chem.,57, 1271, 1974.
255. Coburn, J . A. and Chau, A. S. Y., Confirmation of pesticide residue identity. 1V. Chemical confir-
mation of Dyfonate, Dichlorofenthion and Cyanox, Environ. Lett., 10, 225, 1975.
256. Cochrane, W. P., Confirmation of insecticide and herbicide residues by chemical derivatization, J .
Chromatogr., 13,246, 1975.
257. Bowman, M. C., Holder, C. L., and Rushing, L. G., Trace analysis of etrimfos and two degradation
products in corn and alfalfa, J. Agric. Food Chem., 26, 35, 1978.
258. Singh, J. and Lapointe, M. R., Confirmation of six organothiophosphorus pesticides by chemical
derivatization at nanogram levels, J . Assoc. O f f .Anal. Chem., 57, 1285, 1974.
259. Forbes, M. A., Wilson, B. P., Greenhalgh, R., and Cochrane, W. P., Confirmation of organophos-
phorus insecticides by chemical reduction, Bull. Environ. Cont. Toxicol., 13, 1272, 1975.
260. Greenhalgh, R. and Kovacicova, K., A chemical confirmatory test for organophosphorus and car-
bamate insecticides and triazine and urea herbicides with reactive NH moieties, J . Agric. Food
Chem.,23,325, 1975.
261. Lawrence, J. F. and Iverson, F., Analysis of the diazinon metabolites G 27550 and GS 31 144 by gas-
liquid chromatography with nitrogen-specific detection after derivatization, J . Chromatogr., 103,
341, 1975.
262. Lawrence, J. F. and McLeod, H. A., Gas-liquid chromatographic analysis and chemical confirmation
of azodrin (monocrotophos) residues in strawberries, J . Assoc. O f f .Anal. Chem., 59, 637, 1976.
263. Greenhalgh, R., Dokladalova, J., and Haufe, W. O . , GLC determination of Crufomate (Ruelene)
in bovine blood and the use of U.V. irradiation as a confirmatory test, Bull. Environ. Cont. Toxicol.,
7,237, 1972.
264. Greenhalgh, R.. King, R. R., and Marshall, W. D., Trifluoroacetylation of pesticides and metabolites
containing a sulfoxide moiety for quantitation by gas chromatograph and chemical confirmatory
purposes, J . Agric. Food Chem., 26,475, 1978.
265. Lau, S. C. and Marxmiller, R. L., Residue determination of Landrin insecticide by trifluoroacetyla-
tion and electron-capture gas chromatography, J. Agric. Food Chem., 18,413, 1970.
266. Miller, P. W., Determination of urea in animal tissue by gas chromatography, J . Agric. Food Chem.,
19, 941, 1971.
267. Sieber, J . N., N-perfluoroacyl derivatives for methylcarhamate analysis by gas chromatography, J.
Agric. Food Chem., 20,443, 1972.
268. Khalifa, S. and Mumma, R. O . , Gas chromatographic separation of the aglycone metabolites of
carbaryl, J . Agric. Food Chem., 20,632, 1972.
269. Saunders, G. D. and Vanatta, L. E., Derivatization and gas chromatographic measurement of some
thermally unstable ureas, Anal. Chem.,46, 1319, 1974.
270. Walker, G., Winterlin, W., Fouda, H., and Seiber, J., Gas chromatographic analysis of urethan
(ethyl carbamate) in wine, J . Agric. Food Chem., 22,944, 1974.
271. Wong, L. and Fisher, F. M., Determination of carbofuran and its toxic metabolites in animal tissue
by gas chromatography of their N-trifluoroacetyl derivatives, J . Agric. Food Chem., 23, 315, 1975.
272. Greenhalgh, R., Marshall, W. D., and King, R. R., Trifluoroacetylation of mesurol (4-methylthio-
3.5-xylyl-Nmethylcarbamate), its sulfoxide, sulfone, and phenol analogs by analysis gas chromatog-
raphy, J. Agric. Food Chem., 24, 266, 1976.
273. Greenhalgh, R., Wood, G . W., and Pearce, P. A., A rapid G C method of monitoring mesurol (4-
(methylthio)-3,5,-xylyl-Nmethyl carbamate) and its sulfoxide and sulfone metabolites and their per-
sistence in lowbush blueberries, J. Environ. Sci. Health, 812, 229, 1977.
274. Also see Volume 111, Chapter 1 on carbamate.
275. Woroby, B. L. and Wester, G . R. B., Gas-liquid chromatographic determination of diflubenzuron
(Dimilin) in water as its trifluoroacetyl derivative, J . Assoc. Off. Anal. Chem., 60, 213, 1977.
276. DeMilo, A. B., Terry, P . H., and Rains, D. M., Simple derivatization procedure for gas chromato-
graphic analysis of diflubenzuron in pond water and for analysis of its trifluoromethyl analog penflu-
ron in boll weevils, J. Assoc. Off. Anal. Chem., 61,629, 1977.
277. Bose, R. J . , Formation of the N-trifluoroacetate of carbofuran, J. Agric. Food Chem., 25, 1209,
1977.
278. Larsson, L., The alkaline hydrolysis of two sarin analogues and of tabun, Acta Chem. Scand., 12,
783, 1958.
279. Toy, A. D. F., The preparation of tetraethyl pyrophosphate and other tetraalkyl pyrophosphates, J.
Am. Chem. Soc., 70, 3882, 1948.
280. Toy, A. D. F., Preparation and properties of some unsymmetrical tetraalkyl pyrophosphates, J. Am.
Chem. Soc., 72,2065, 1950.
281. Green, A. L., Sainsbury, G . L., Saville, B., and Stanfield, M., The reactivity of some active nucleo-
philic reagents with organophosphorus anticholinesterases, J. Chem. Soc., p. 1583, 1958.
282. Fukuto, T. R. and Metcalf, R. L., Isomerization of /3-ethylmercapto-ethyl diethyl thionphosphate
(Systox), J. Am. Chem. Soc.,76,5103, 1954.
283. Eto, M., Organophosphorus Pesticides: Organic and Biological Chemistry, CRC Press, Boca Raton,
Fla., 1974, 26 and 90.
283b. Eto, M., Organophosphorus Pesticides: Organic and Biological Chemistry, CRC Press, Boca Raton,
Fla., 1974, 89.
284. Hilgetag, G. and Teichmann, H., The alkylating properties of alkyl thiophosphates, Angew. Chem.
Int. Ed.,4,914, 1965.
285. Lawrence, J . F., Evaluation and confirmation of an alkylation-gas-liquid chromatographic method
for the determination of carbamate and urea herbicides in foods, J. Assoc. Off. Anal. Chem., 59,
1061, 1976.
286. Buchert, A., and L+kke, H., Gas chromatographic-mass spectrometric identification of phenylurea
herbicides after N-methylation, J. Chromatogr., 115,682, 1975.
287. Greenhalgh, R. and Kovacicova, J., Confirmation of atrazine and fenuron by alkylation at p.p.m.
level, Bull. Environ. Cont. Toxicol., 14, 47, 1975.
288. Lawrence, J. F. and Lavar, G . W., Analysis of some carbamate and urea herbicides in food by gas-
liquid chromatography after alkylation, J. Agric. Food Chem., 23, 1106, 1975.
289. Lawrence, J . F., Lewis, D., and McLeod, H. A., Confirmation of some NO,-containing pesticides
by chemical reduction and gas chromatography with electrolytic conductivity detection, J. Agric.
Food Chem., 25, 1359, 1977.
290. Forbes, M. A., Confirmation of parathion, fenithrothion and methyl parathion in residues using
chromous chloride, in-house report, Water Quality Branch, Environment Canada, Moncton, N.B.,
June 1973.
291. Ripley, B. D., Hall, J . A., and Chau, A. S. Y., Determination of fenitrothion, phosphamidon and
dimethoate in natural water, Environ. Lett., 7,97, 1974.
292. Cochrane, W. P. and Purkayastha, R., Analysis of herbicide residues by gas chromatography, Tox-
icol. Environ. Rev., 1, 137, 1973.
293. Khan, S. U., Chemical derivatization of herbicide residues for gas liquid chromatographic analysis,
Res. Rev., 59, 22, 1975.
294. Yip, G . , Analysis of herbicides and metabolites, J. Chromatogr. Sci., 13, 225, 1975.
295. Chau, A. S. Y. and Terry, K., Analysis of pesticides by chemical derivatization. I. A new procedure
for the formation of 2-chloroethyl esters of ten herbicidal acids, J. Assoc. Off. Anal. Chem., 58,
1294, 1975.
296. Chau, A. S. Y. and Terry, K., Analysis of pesticides by chemical derivatization. 111. Gas chromato-
graphic characteristics and conditions for the formation of pentafluorobenzyl derivatives of ten her-
bicidal acids, J. Assoc. Off. Anal. Chem., 59,633, 1976.
297. Agemian, H. and Chau, A. S. Y., Determination of pesticides by derivative formation. IV. A sensi-
tive gas-chromatographic method for the determination of MCPA and MCPB herbicides after ester-
ification with I-bromoethyl-2,3,4,5,6,-pentafluorobenzene Analyst, 101, 732, 1976.
Volume I 171
298. Agemian, H. and Chau, A. S. Y., Analysis of pesticide residues by chemical derivatization. V. Mul-
tiresidue analysis of eight phenoxyalkanoic acid herbicides in natural waters, J. Assoc. Off. Anal,
Chem., 60, 1070, 1977.
299. Larose, R. H. and Chau, A. S. Y., Herbicide analysis: relationship between molecular structure and
retention index, J. Assoc. Off. Anal. Chem.,56, 1183, 1973.
300a. Sattar, M. A,, Hattula, M. L., Lahitipera, M., and Paasivirta, J., Improved analysis method of
MCPA from soil samples, Chemosphere, 11, 747, 1977.
300b. Sattar, M. A. and Paasivirta, J., Simultaneous determination of 4-chloro-2-methyl phenoxyacetic
acid and its metabolites in soil by gas chromatography, Anal. Chem., 51, 598, 1979.
301. Cotterill, E. G., Rapid simultaneous determination of residues of MCPA, mecoprop and MCPB in
soil by gas chromatography of the pentafluorobenzyl ester, J. Chromatogr., 171,478, 1979.
302. Gutenmann, W. H. and Lisk, D. J., Rapid method for MCP in soil by electron affinity determination
of 2-chloroethyl ester, J. Assoc. Off. Anal. Chern.,47,353, 1964.
303. Woodham, D. W., Mitchell, W. G., Loftis, C. D., and Collier, C. W., An improved gas chromato-
graphic method for the analysis of 2,4-D free acid in soil, J. Agric. Food Chem., 19, 186, 1971.
304. Van Peteghem, C. and Heyndrickx, A., Identify confirmation of chlorophenoxyacid herbicides resi-
dues, Mededel. Fac. Lanbouw. Gent.,38,857,1973.
305. Thier, H-P., Analysis of herbicides, Angew. Chem. Int. Ed., 13,217, 1974.
306. Gutenmann, W. H. and Lisk, D. J., Preparation of electron capturing derivatives of insensitive
pesticides, J. Assoc. Off. Agric. Chem.,46, 859, 1963.
307. Sherma, J., Manual of Analytical Quality Control for Pesticides and Related Compounds in Human
and Environmental Samples, EPA-600/1-76-017, U.S. Environmental Protection Agency, Research
Triangle Park, N.C., 1976.
308. Glad, G., Popoff, T., and Theander, O., Determination of liuron and its metabolites by GLC and
HPLC, J. Chrornatogr., 16, 118, 1978.
309. Magallona, E. D., Gas chromatographic determination of residues of insecticidal carbamates, Res.
Rev., 56, 1, 1975.
310. Hall, R. C. and Harris, D. E., Direct gas chromatographic determination of carbamate pesticides
using Carbowax 20M-modified supports and the electrolytic conductivity detector, J. Chrornatogr.,
169.245, 1979.
31 1. Cochrane, W. P. and Purkayastha, R., Analysis of herbicide residues by gas chromatography, Tox-
icol. Environ. Chem. Rev., l , 137, 1973.
312a. Wales, P. and Mendoza, C. E., Investigation on the determination and confirmation of dyrene added
to plant extracts: GLC and TLC of Dyrene and products of its reaction in methanolic sodium hy-
droxide, J. Assoc. Off. Anal. Chem.,53, 509, 1970.
312b. Mendoza, C. E., Wales, P. J., and Hatina, G. V., Alkoxy derivatives of Dyrene. Identification and
carboxylesterase inhibition, J. Agric. Food Chem., 19, 41, 1971.
313. Lawrence, J. F., Confirmation techniques for triazine herbicides by gas chromatography with elec-
trolytic conductivity detection, J. Agric. Food Chem., 22, 936, 1974.
314. Cochrane, W. P. and Greenhalgh, R., Confirmation of pesticide identity by chemical derivitization,
Abstracts of Papers, 166th Natl. Meeting of the American Chemical Society, Chicago, August 26 to
31,1973.
315. Khan, S. U., Greenhalgh, R., and Cochrane, W. P., Chemical derivitization of hydroxyatrazine for
gas chromatographic analysis, J. Agric. Food Chem., 23,430, 1975.
316. Coward, R . F. and Smith, P., The detection of nitrogenous compounds using a thermionic detector,
J. Chrornatogr., 61, 329, 1971.
317. Cee, A. and Gasparic, J., Nachweis der sTriazin-Gruppe in organischen Verbindungen. LXXVIII.
Mitt. uber die identifizierung organischer Verbindungen, Mikrochim. Acta, p. 823, 1972.
318. Flint, G. T. and Aue, W. A., The gas-liquid chromatographic determination of 2-hydroxy-striazines,
J. Chromatogr., 52,478, 1970.
319. Montgomery, M. L., Botsford, D. L., and Freed, V. H., Metabolism of hydroxy-simazine by corn
plants, J. Agric. Food Chem., 17, 1241, 1969.
320. Sirons, G. J., Frank, R., and Sawyer, T., Residues of atrazine, cyanazine, and their phytotoxic
metabolites in a clay loam soil, J. Agric. Food Chem., 21, 1016, 1973.
321. Tadic, Z. D. and Ries, S. K., Thermal and ultrasonic dealkylation of S-triazines via the cyclic transi-
tion state, J. Agric. Food Chern., 19, 46, 1971.
322. Plimmer, J. R., Kearney, P. C., and Klingebiel, U. I., S-Triazine herbicide dealkylation by free-
radical generating systems, J. Agric. Food Chem., 19,572, 1971.
323. Schroeder, R. S., Patel, N. R., Hedrich, L. W., Doyle, W. C., Riden, J. R., and Phillips, L. V.,
Analytical method for determination of residue levels of hydroxycyprazine (2-hydroxy-4-cyclopro-
pylamino-6-isopropylamino-striazine) in corn, J. Agric. Food Chern., 20, 1286, 1972.
324. Lau, S. C., Katague, D. B., and Stoutamire, D. W., Characterization and microdetermination of a
water-soluble metabolite from Bladex herbicide by conversion to 5.5-dimethylhydantoin, J . Agric.
Food Chem., 21, 1091, 1973.
325. Drozd, J., Chemical derivatization in gas chromatography, J. Chromatogr., 113,303,1975.
326. Ahuya, S., Derivatization in gas chromatography, J. Pharmacol. Sci., 65, 163,1976.
327. Lawrence, J. F. and Frei, R . W., Chemical Derivatization in Liquid Chromatography, Elsevier,
Amsterdam, 1976.
328. Blau, K. and King, G. S., Eds.,Handbook of Derivatives for Chromatography, Heyden and Sons,
London, 1977.
329. Beroza, M. and Bowman, M. C., Instrumentation in determination of organophosphorus terminal
residues, in Pesticide Terminal Residues, Tahori, A. S., Ed., Butterworth, London, 1971.
330. Afghan. B. K., Organic Analysis of Water Pollution: Chemical Analysis, CRC Press, Boca Raton,
Fla., in press.
Volume I 173
Chapter 4
THE CHEMISTRY OF THE CYCLODIENE INSECTICIDES
John W .ApSimon and Kazuyuki Yamasaki
TABLE OF CONTENTS
Introduction ......................................................174
A Note on Nomenclature ...........................................174

Synthesis .........................................................177
A. Diene ......................................................177
B. Hexachlorocyclopentadiene ...................................179
C. Chlordane ..................................................179
D. Heptachlor .................................................180
E. Aldrin and Dieldrin ..........................................180
F. IsodrinandEndrin ...........................................180
Chemical Reactions of Heptachlor. Chlordane. and Related Compounds ... 181
Chemical Reactions of Aldrin. Isodrin. and Related Compounds .......... 183
Chemical Reactions of Dieldrin. Endrin. and Related Compounds . . . . . . . . . 189
Summary .........................................................194
Appendix -- The Wagner-Meerwein Rearrangement ....................194

Acknowledgments .................................................194
References ..............................................................195
I. INTRODUCTION
The cyclodiene insecticides constitute a series of chlorinated hydrocarbons that are

produced commercially in order to cope with insects and pests against grains and crops.
Their discoveries date back to the 1940s, and they have been widely used for insect
control as the most effective insecticides known to date. During these years, the usage
of these insecticides has raised serious controversies due to their persistent nature in
the ecosystem and is now partly forbidden or limited. In view of the possible environ-
mental degradation caused by the residue problem of the insecticides, it is desirable
that their behavior in the ecological system be more understood so that the degradation
products formed by metabolic and environmental conversions of the insecticides can
be analyzed and their effects upon living organisms, especially animals and human
beings, can also be estimated. In this sense, the understanding of the chemical changes
of these substances appears to be of significant importance since changes in vitro fre-
quently parallel or bear a close resemblance to those which might be expected to occur
in vivo. For these reasons and from the chemical point of view, the chemistry of the
cyclodiene insecticides has attracted considerable attention. Consequently, it is not sur-
prising that the chemistry itself has been reviewed more than a few times in the past,
appearing in the review of Ungnade and McBee in 1958,' a brief report by Riemschnei-
der that appeared in 1963,' a Russian version by Menikou, whose translated article
was published in Residue review^,^ and more recently the comprehensive review by
Brooks in 1974.4 Additionally, a review focused on the photochemical reactions of the
insecticides has been published by Korte and Parlar.'
Since the above-mentioned reviews cover most of the chemical aspects of the insec-
ticides and, moreover, it is not feasible to produce a more comprehensive outlook on
the matter than Brooks' excellent work, this review is necessarily limited to a survey
of some aspects involved in the chemical transformations of the insecticides. Since the
photochemistry of the insecticides is treated independently in another chapter, it will
not be included here so as to avoid the duplication of material; instead, a brief treat-
ment of the synthesis of the insecticides is presented to familiarize readers with their
structural characteristics. It is recommended that those who are interested in such de-
tails as the actual processes by which the insecticides are prepared on the industrial or
laboratory scale, the composition of the commercial products, the history of their de-
velopment, the present status of usage and so on should consult the afore-mentioned
reviews. The chlorohydrocarbons which are dealt with in this review appear in Fig-
ure 1 as indicated in the Formulas l , 2, 3 , 4 , 5 , 6 and 7 under their accepted names.
11. A NOTE ON NOMENCLATURE
One difficulty that most readers who are not familiar with the insecticides encounter
at first sight of these molecules is the nomenclature. The systematic naming as well as
the numbering system of these molecules creates some confusion among those who
lack a thorough understanding of the systems for the naming of organic compounds.
The present situation is complicated even more so by the isolation of complex cage
molecules derived from molecular rearrangements of these insecticides. Therefore, for
the convenience of readers, it is reasonable to give a brief description of the nomencla-
ture of the chlorohydrocarbons listed in Figure 1 before we proceed with the exami-
nation of their chemical properties. As it is beyond the scope of this review to treat
the matter thoroughly, it is suggested that the Brooks review (page 87)4 be consulted
for a detailed description on nomenclature in general and the naming procedure for
the insecticides.
For the sake of convenience the accepted names given in Figure 1 will be used
Aldrin lsodrin
(41 (5)
FIGURE 2
After having completed the operations above, the full correct name of heptachlor
emerges as 1-exe4,5,6,7,8,8-heptachloro-3a,4,7,7a-tetrahydro-4,7-methanoindene. In
a similar fashion, cischlordane (1) is named as l-exo, 2-exe4,5,6,7,8,8-octachloro-
2,3,3a,4,7,7a-hexahydro 4,7-methanoindene, and "l-exo, 2-endow is simply attached
in place of "l-exo, 2-exoas a prefix for transchlordane.
In this system of nomenclature, aldrin (4) and isodrin (5) can be regarded as arising
from napthalene (1 1). Accordingly, similar operations to those described in Scheme 1
can also be applied to aldrin and isodrin, and the planar structure (12) of both sub-
stances results (Scheme 2). The planar structure (12) can be read as 1,2,3,4,10,10-hex-
achloro-1,4,4a,5,8,8a-hexahydro-l,4; 5,8-dimethanonapthalene.
SCHEME 2
In order to distinguish aldrin from isodrin, the prefix "endo" or "exo" is attached as
an addendum of the planar structure. According to Brooks explanation (see Reference
4, p. 91) based on the previous suggestion6 the terms "exo" and "endo" are defined
in such manner that they imply which of the pairs of exo or end0 bonds of the norbor-
nene nuclei are used to fuse the two rings together. Accordingly, the term "1,4-end*
5,8-exo" is applied to aldrin; on the other hand, "1,4-ende5,8-endo" is to be utilized
for isodrin. The relationship of one ring to the other can be understood as shown in
Figure 2.
Taken together, the full name for aldrin is therefore 1,2,3,4,10,10-hexachloro-
1,4,4a,5,8,8a-hexahydro-l,4-endo,5,8-exo-dimethanonapthalene. The name,
1,2,3,4,10,10-hexachloro-l,4,4a,5,8,8a-hexahydro-l,4-endo, 5,8-endedimethanonap-
thalene, is given to isodrin.
Following this nomenclature, an epoxide fused to norbornene ring can be similarly
added to the name of the parent molecule by denoting exeepoxy or e n d e depending
upon its mode of attachment. Thus, as a logical extension, dieldrin (6) is named as
1,2,3,4,10,10-hexachloro-6,7-exeepoxy-l,4,4a,5,6,7,8,8a-octahydro-l,4-endo, 5,8-
exedimethano-napthalene. Endrin (7) is 1,2,3,4,10,10-hexachloro-6,7-exeepoxy-
1,4,4a,5,6,7,8,8a-octahydro-l,4-endo, 5,8-endedimethanonapthalene.
Although the nomenclature presented above seems quite logical and convenient in
describing the insecticides that can be correlated to aromatic hydrocarbons, it becomes
inadequate or even inapplicable when it comes to the more complex ring structures
produced by various molecular rearrangements. In this connection, another systematic
nomenclature for naming polycyclic hydrocarbons which is more accurate and of wide
versatility has been proposed by Eckroth7 based on correct von Baeyer names (IUPAC
1957 rule^).^ Bensong has subsequently applied this nomenclature to the cyclodiene
insecticides.
FIGURE 3
In the Eckroth system, the structure of a molecule is first drawn in a planar graph
(called a Schlegel diagram) and the largest possible circuit of the planar graph is deter-
mined and is numbered t o satisfy the IUPAC rules. This system has proved to be very
useful for adoption for the uniform naming of aldrin, dieldrin, etc. For example, diel-
drin can be drawn in a planar graph (Figure 3). This planar graph provides the follow-
ing name for dieldrin, assuming C-l and C-8 are the two key bridgehead positions that
separate three major rings, ring C-1-1 1-8, ring C-1-10-9-8, and ring C-1-2-3-4-5-6-7-8:
1,8,9,10,11,11-hexachloro-4,5-exeepoxy-tetracyclo [.2.1.1 .3.6.02.7]dodec-9-ene. For
the same reason as mentioned earlier to clarify the difference between aldrin and is-
odrin structure, "2,3-7,6-ende2,l-7,8-exo" should be added to the name above; thus
the final name of dieldrin, according to this nomenclature, is as follows:
1,8,9,10, l l , l l-hexachloro-4,5-exo-epoxy-2,3,-7,6-endo-2,1-7,8-exetetracyclo
[6.2.1. 13.6.02.7]dodec-9-ene. Similarly endrin is named as follows: 1,8,9,10,11,1 l-hex-
achloro-4,5-exeepoxy-2,3-7,6-ende2,1-7,8-endetetracyclo [6.2.1.13.6.02.7] dodec-9-
ene. The full correct names of aldrin and isodrin would be derivable if the term "4,5-
exeepoxy" were deleted from the corresponding names for dieldrin and endrin.
111. SYNTHESIS
A. Diene
As is evident from Figure 1, the cyclodiene insecticides that will be discussed in this
review are highly chlorinated polycyclic compounds, consisting of a hexa-chlorinated
molecular portion and a less or nonchlorinated portion with an extra double bond or
epoxide. The key reaction used to prepare these compounds is called the Diels-Alder
reaction. This reaction is so well known that any textbook on organic chemistry usually
allocates an independent chapter for describing the reaction, and there are a number
of monographs on the subject.lO~"Considering the importance played by the reaction
in the preparation of the insecticides, a brief account on some general features of the
Diels-Alder reaction is presented in this section.
In general, the Diels-Alder reaction is comprised of a reaction between two compo-
nents, a diene and dienophile, with concommitant formation of a six-membered ring
as shown in Scheme 3.
SCHEME 3
It is well known that the presence of electron-donating substituents (CH,, OCH,,

C6H,, etc.) in the diene molecule increases its reactivity, while electron-withdrawing
substituents (COOH, CN, etc.) decrease it. By contrast, electron-withdrawing substi-
tuents conjugated with the reacting double or triple bond of the dienophile accelerate
ring formation when compared to the case in which the dienophile is not activated.
Dienophiles can also be compounds possessing multiple bonds of the type C=O, C=N,
N=O, etc., in which case the resulting adducts are heterocyclic compounds. Thus, the
diene synthesis has become a very versatile method for the construction of polycyclic
systems, both carbocyclic and heterocyclic, and has been used widely for this purpose.
There are two important stereochemical features that should be pointed out regard-
ing the formation of the Diels-Alder adduct. The first one, known as Alder's rule,'' is
that the diene molecule adds to the double bond (or triple bond) so as to produce a
cisrelationship with respect to the newly formed bonds, while maintaining the relative
spatial relationship of substituents in the dienophile through the course of addition.
This mode of addition can be best demonstrated in the following example (Scheme 4).
":co >- (JJJH:

.
p,
,,OOCd//-OOHU
>
'COOH
SCHEME 4
Thus, the Diels-Alder reaction between butadiene and maleic anhydride gives rise to
the formation of the cis-fused adduct (13), while the transsubstituted adduct (14) is
obtainable from butadiene and fumaric acid.
The other significant stereochemical outcome observed in the Diels-Alder addition
is the predominant formation of an endeisomer. This result follows the rule that the
both components (diene and dienophile) orient themselves in an end0 fasion, in the
transition state and that an endeisomer is principally produced as the result of this
orientation. This rule can be explained by the assumption that the molecules tend to
arrange themselves in the transition state where the maximum n-electron overlap can
be attained.
c@-isomer CXO- isomer

(prefered isomer)
SCHEME S
Although two orientations in the transition state for the addition between cyclopenta-
diene and maleic anhydride are possible as illustrated by Scheme 5 by which an e n d e
or exeisomer results, respectively, only endeaddition is normally seen. According to
a more modern interpretation proposed by Woodward and Hoffman,13 it is postulated
that the endeapproach is favored over the e x e as the result of secondary orbital in-
teractions which contribute to lower the energy state of the endetransition state com-
pared to that of the exetransition state unless the addition is governed by special
factors such as secondary isomerization or steric control that might alter the course of
addition. In any event, regardless of what the rationalization may be, it is now possible
to predict the structure of a Diels-Alder adduct including its stereochemistry.
B. Hexachlorocyclopentadiene
Hexachlorocyclopentadiene is undoubtedly located as a key compound for the syn-
thesis of cyclodiene insecticides. The Diels-Alder adducts from this compound have
proved to be active in insecticidal properties, some of which are currently used as
commercial insecticides.
The first published method for the preparation of hexachlorocyclopentadiene ap-
peared in 1930,14and consisted of the chlorination of cyclopentadiene (15) in alkaline
hypochlorite (Scheme 6).
SCHEME 6
The other industrial method consists of the chlorination of a C, hydrocarbon mixture

conducted at high temperature, during which process2 hydrocarbons are chlorinated
photochemically until they have an average composition of C,H,CI,.
The ability of hexachlorocyclopentadiene to participate in the Diels-Alder reaction
was first illustrated by PrillI5 in 1947 and since that time numerous examples of the
cyclo adducts resulting from (16) have appeared. The fact that hexachlorocyclopenta-
diene is an excellent partner in the Diels-Alder addition is mainly ascribed to the ther-
mal stability of the diene itself, for which reason it does not form dimers or polymers
under the usual reaction conditions.
C . Chlordane
The first insecticide of the diene group became known by the name of chlordane,
and its main component is the chlorohydrocarbon (17). The planar structure for this
substance is shown together with the numbering system. Among four possible stereo-
isomers representing structure (17), only two have been shown to exist in technical
chlordane as major components; these are the l-exo,2-exo (or cis) (1) and the 1-
e x e , 2 e n d e (or trans)(2) isomer, respectively. The terms exo or end0 refer in this case
to the orientation of the chlorine atom being on the exo or end0 side of the parent
molecule. The stereostructures of both cis and trans chlordanes have already been
given in Figure 1.
The Diels-Alder adduct (18) derived from hexachlorocyclopentadiene (16) and cyclo-
pentadiene (15) is the starting material for the preparation of commercial chlordane
and heptachlor (Scheme 7).
SCHEME 7
Adduct (18) is then chlorinated with sulfuryl chloride in the presence of aluminium
chloride or with chlorine in carbon tetrachloride, producing a mixture of products with
the average composition of CloHsCls' corresponding to commercial chlordane which
is a complex mixture of chlorinated hydrocarbons besides (1) and (2) varying in insec-
ticidal activity.
D. Heptachlor
Heptachlor is a similar insecticide to chlordane (17) and is prepared by direct chlo-
rination of chlordene (18) with sulfuryl chloride in carbon tetrachloride in an analo-
gous manner to the preparation of chlordanel or by a two-step process16which involves
allylic oxidation with selenium dioxide in the presence of acetic acid [(18) (19)] fol-
+
lowed by replacement of the acetyl group by chlorine as shown in Scheme 8.
(181 -> Cl
c ' ~ o cHCI o> cL31 ~
Cl
SCHEME 8
The main component of the commercial heptachlor is the chlorohydrocarbon (3).
E. Aldrin and Dieldrin

In the early 1950s the discovery of two new i n s e c t i ~ i d e s , "aldrin
~ ~ ~ and dieldrin, was
announced. These insecticides were found to be more effective than any other known
chlorinated insecticides at that time.
Norbornadiene (20) is employed as a dienophile in this instance, which readily com-
bines with (16) to yield an adduct (4). In agreement with the endeaddition rule for
the orientation of the Diels-Alder reaction, the major product was shown to be com-
pound (4). The steric course of this addition reaction as well as the stereostructure of
the product is depicted in Scheme 9.
SCHEME 9
Aldrin is the accepted name for compound (4), and the oxidation of (4) with hydro-
gen peroxide o r with peracid (peracetic acid or perbenzoic acid) gives dieldrin (6).
F. Isodrin and Endrin

Isodrin and endrin are the corresponding stereoisomers of aldrin and dieldrin which
were developed a few years after the discovery of aldrin and dieldrin.".*O
Hexachlorobicyclo [2,2,1] heptadiene (22) is introduced as a key component in the
synthesis. Thus Diels-Alder reaction of (16) with vinyl chloride yields an adduct (21).
which in turn is converted by dehydrochlorination (Scheme 10) into the hexachloron-
orbornadiene (22).
Volume I 181
SCHEME 10
A second Diels-Alder reaction of (22) with (15) results in isodrin (S), an isomer of
aldrin ( 4 ) in a similar fashion to the aldrin synthesis (Scheme 11).
SCHEME 1 1
Endrin is ( 7 ) derived from isodrin by epoxidation.
IV. CHEMICAL REACTIONS OF HEPTACHLOR, CHLORDANE, AND

RELATED COMPOUNDS
The general features of the chemical reactions of chlordane and related compounds
are chiefly centered on the cyclopentane portion of the molecules. Reduction of hep-
tachlor ( 3 ) in acetone with aqueous chromous chloride solution yields the pentachloro
derivative (23) eventually, but the reaction is found to proceed in two steps (Scheme
12).21The first stage is much faster than the second reduction, and the same compound
(23) can be obtained from Zn-AcOH reduction of chlordene (18) and heptachlor ( 3 )
albeit in a lower yield.
SCHEME 12
When heptachlor ( 3 ) is treated with alcoholic sodium methoxide, it is converted t o

(24) in which chlorine at C - l has been simply replaced.z2 Under more drastic condi-
tions, at 110 to 130°C and under 12 kbar, solvolysis of ( 3 ) in n-butanol with a catalytic
amount of sodium hydroxide produces princiaplly (24) and (25) in addition to small
amounts of (26) and ( 2 7 ) , the last two compounds being derived from the addition of
either n-butanol o r hydrogen chloride to the 2,3-double bond.23These transformations
are summarized in Scheme 13.
SCHEME 13
When heptachlor-2,3-epoxide (28), a biological metabolite of heptachlor," is treated

with boiling methanolic sodium methoxide, compound (29) is formed, which in turn
gives (30) on treatment with thionyl chloride." Compound (30) can be reconverted to
(29) by boiling in methanolic silver carbonate. These conversions are displayed in
Scheme 14. A concerted mechanism involving the initial abstraction of the C-l hydro-
gen by base and synchronous ring-opening of the epoxide has been postulated for this
base-induced epoxide cleavage (Scheme 14). A transcoplanar arrangement of the hy-
drogen being abstracted (C-l endehydrogen) and the 2,3-epoxide ring is apparently
92>vl
the factor facilitating this transformation.
MeOH >
NoOCH
Ae2co,
(3' Cl in oq. MeOH
B
SCHEME 14
It is interesting to compare the above results with the behavior of the isomeric end0
epoxide (31) under the same conditions. A similar reaction takes place when (31) is
treated with the base, giving this time the isomeric allylic alcohol (32).26In this in-
stance, the initial hydrogen abstraction must occur at the C-3a position rather than at
C - l , since the former hydrogen is trans to the epoxide, while the latter is cis to the
epoxide as shown in Scheme 15. As in the above example [i.e., (29) -* (30)], the hy-
droxy group at C-2 of (32) can be replaced easily by chlorine on treatment with thionyl
chloride. Oxidation of (32) with active manganese dioxide gives the keto compound
(34), but on boiling (33) with aqueous alcoholic silver carbonate no reconversion to
(32) is observed [cf. (30) -* (29)l.
SCHEME 15
Volume I 183
The fact that transepoxide ring-opening which is commonly observed does not oc-
cur in both these cases is worthy of comment. In order to fulfil1 the steric requirements
for the transepoxide ring-opening, reagents must attack the epoxide ring from its rear
side. As far as this condition is concerned, it can be said that steric hindrance due to
the endeconfiguration of the exeepoxide (28) precludes the approach from the rear
face in (28) and that the chlorine atom at C -1 probably inhibits the rear-side attack in
the case of (31).
The situation with cis and transchlordanes, (1) and (2). is also interesting; the same
base treatment as previously mentioned induces dehydrochlorination in both com-
pounds (1) and (2).22It is found that the rate of the dehydrochlorination of trans(2)
is much slower than the corresponding rate observed in cis-(l), the products being (35)
and (36), respectively. A stereochemical requirement similar to those discussed in the
foregoing examples seems to be operating again whereby the abstraction of the proton
at C-3 in (1) is much more favorable than that at C-3 in (2), allowing the elimination
to proceed in a more readily in a transfashion compared to (2) which is only capable
of ciselimination, as illustrated in Scheme 16.
SCHEME 16
V. CHEMICAL REACTIONS OF ALDRIN, ISODRIN, AND RELATED

COMPOUNDS
The chemical reactions of aldrin and isodrin may be divided into the following three
types: (1) addition reactions, (2) dehalogenation, and (3) transannular reactions.
In the first category, it has been shown that electrophiles, radicals, and dienes can
undergo addition reactions in normal sense to the unchlorinated double bond of these
molecules. The trichloromethyl radical generated either from chloroform or carbon
tetrachloride with the aid of benzoyl peroxide adds to the 6,7-double bond of aldrin,
and the products are (37) and (38) depending on the radical precursor (Scheme 17).27
Compound (38) is identical with the product obtained from chloroform and 6-chlo-
roaldrin.
SCHEME 17
The exeattack of the radical was assumed based on the earlier reports on ionic and
radical additions to norborene type compounds. A similar addition of l-iodoperfluo-
ropropane t o aldrin has been recorded.28Treatment of aldrin with a solution of phen-
ylazide in hexane affords the phenyltriazole derivative (39) after evaporation of the
solvent and heating the residue at 85°C (Reference 4, p. 132). pToluenesulfonyl azide
reacts with isodrin to give the aziridine derivative (40), no intermediate dihydrotriazole
such as (39) being detected in this i n ~ t a n c e . ' Compound
~ (40) undergoes a series of
transformations that are related to those of endrin which will be discussed in the next
section, one of them leading to the cage amine (41) in hot trifluoroacetic acid. These
azide additions are outlined in Scheme 18.
SCHEME 18
Oxidation of aldrin and isodrin with alkaline potassium permanganate or osmium

tetroxide gives cis-diols (42) and (43), c o r r e ~ p o n d i n g l y Since
. ~ ~ both (42) and (43) form
their corresponding acetonides (44) and (45) when treated with acetone in the presence
of ferric chloride, the cisrelationship of the two hydroxy groups is secured. The hy-
droboration-oxidation procedure converts aldrin (4) to the exealcohol(46). Oxidation
of (46) with sodium dichromate in sulfuric acid gives ketone (47). The stereoselective
reduction of the ketone (47) with disiamyl borane affords the endealcohol (48), and
it is reconverted to (47) by oxidation with chromic acid. The relationship of the com-
pounds derived from aldrin and isodrin is delineated in Scheme 19. Compounds (42),
(46), and (47) are metabolites of aldrin in plant^.^'
SCHEME 19
Volume I 185
Although aldrin and isodrin are inert towards various reducing reagents such as
sodium borohydride, potassium borohydride, or lithium aluminium hydride, they react
with sodium borohydride in the presence of Co2+ion [ C O ( N O ~. 6) ~ H 2 0 ]to give dechlo-
rinated compounds (49) and (50), r e ~ p e c t i v e l yThis
. ~ ~ reagent stereoselectively removes
the syn-chlorine in both aldrin and isodrin (Scheme 20).
SCHEME 20
It was also believed that the chlorinated hydrocarbons were stable enough to survive
under a wide range of basic reagents, and this stability was considered to be due to
steric inhibition to nucleophilic attack in S,2 reactions or to carbonium ion formation
to allow S,1 reactions. However, MacKenzie and ad am^^^ have reported that the ster-
eospecific dechlorination of these hydrocarbons can take place on treatment with al-
koxide ion. Thus, the mono-dechlorinated hydrocarbon (51) forms from aldrin by
heating with sodium methoxide in methanol-dimethyl sulfoxide. A similar reaction
with isodrin gives its corresponding dechlorinated hydrocarbon (52). This reagent is
found to eliminate the antgchlorine selectively (Scheme 21).
SCHEME 21
A number of interesting transannular reactions have been discovered in the isodrin

series as the proximity of the two double bonds permits mutual interaction upon attack
of electrophilic reagents. This is in marked contrast to aldrin, which normally under-
goes simple addition reactions. The following reactions of aldrin and isodrin specifi-
cally demonstrate this point.34 Acetic acid containing sulfuric acid turns aldrin almost
exclusively into the acetate (53) with a detectable amount of the half-cage acetate (54)
(whose origin will be discussed later), while under the same conditions isodrin is con-
verted into (54). The initially assumed structure (55) for this acetate was revised to
structure (54) on account of the absence of the strong IR absorption around 1600 cm-l
(Cl-C=C-Cl). Oxidation of the alcohol (56) derivable from (54) with potassium per-
manganate gives the ketone (57) identical with that produced from endrin with boron
trifluoride in benzene (57).35In another instance, isodrin when treated with either HBr
or Br, in carbon tetrachloride gives mainly the cage product (58). It has been already
shown that UV irradiation of isodrin yields the same saturated product (58).36These
transformations are summarized in Scheme 22.
SCHEME 22
The formation of the half-caged acetate (54) from isodrin can be explained as follows:
(1) first a carbonium ion (59) with the charge on C-7 is formed by attack of the C-6,7
double bond by a proton; (2) a pair of electrons is shifted from the C-2,3 double bond
to form a new bond between C-2 and C-7 with a new positive charge being created on
C-3 (60); (3) a hydride shift then occurs from C-6 to C-3 and the resulting carbonium
ion (61) is quenched by acetate ion, leading to (54). These steps are summarized in
Scheme 23.
SCHEME 23
In the formation of the cage product (58) ion pairs such as (62) or (63) may be involved,
and the product can probably arise from either of them by removal of Br' by Br- in
preference t o H'.
Brooks4 ( p l . 138) proposed an alternative mechanism [as shown in structure (64)l in

which two electrons from the C-2,3 double bond form a new bond between C-2 and
C-7, and then the resulting carbonium ion on C-3 is discharged by transannular linking
between C-3 and C-6 with extrusion of Br'.
Volume 1 187
MacKenzie3' examined the behavior of the chlorohydrocarbon (65) under similar

reaction conditions t o those described above. Thus, (65) is found to undergo transan-
nular reactions, with a few interesting differences in the results. Bromination of (65)
with bromine-acetic acid affords a bromoketone (66), but mineral acids (HBr and
AcOH, H2S04) transform (65) t o a saturated ketone (67) (Scheme 24).
SCHEME 24
The ketones, (66) and (67), are formed by way of the intermediate carbonium ions,
(68) and (69), which are transformed into (70) and (71), respectively, by the same proc-
ess as outlined in Scheme 23 [i.e., (59) + (61)l. The ions (70) and (71) capture the
solvent in this case, which is different from the change (60) -,(61), the products (72)
and (73) decomposing eventually to ketones (66) and (67). This mechanism is illustrated
in Scheme 25.
l
(66) and (671
SCHEME 25
Although the foregoing examples seem to give the impression that aldrin had little
propensity for skeletal rearrangement under any circumstances, there have been, in
fact, a limited number of (but yet significant) instances which demonstrate that skeletal
rearrangements take place in aldrin derivatives. In this context, Mackenzie and co-
have reported recently that monochloroaldrin (5 l ) and monodechloroisodrin
(52) are both rearranged to give the common ketone (74) (Scheme 26). The synthesis
of these substances from their parent chlorohydrocarbons has been mentioned
earlier.33 Isodrin is recovered unchanged from neat sulfuric acid, but stirring (51) or
(52) with sulfuric acid gives in each case the same ketone (74) and several minor prod-
ucts. Structure (74) was secured on the basis of the detailed analysis of its NMR spec-
trum. When direct recrystallization is effected in place of column chromatography
(silica gel) of the crude reaction mixture, the isomer of (74), (75), is only isolable as a
major product. Additionally, (75) is quantitatively converted to its isomer (74) on pas-
sage through a silica gel column.
5 "SO
4>
(52) Silica gel
SCHEME 26
In any case, the formation of the same ketone from both aldrin and isodrin type struc-
tures requires an intermediacy of a common precursor; therefore, the mechanism
shown in Scheme 27 has been proposed to account for this rearrangement. The mech-
anism here implies that the ketone (75) is derivable from (51) through two Wagner-
Meerwein 1,2-sigmatropic shifts [(76) (77) and (78) -* (75); see Appendix] with the
+
intervention of a transannular ring closure in (77) whereas that for (52) transannular
ring closure and a single 1,Zshift are required. In neat sulfuric acid, equilibrium is
probably achieved among the cations [(76), (77), and (78)] due to the poor nucleophilic
power of the medium; thus the product is predominantly derived from the thermody-
namically most stable cation species [probably (78) in this case]. The mechanism pre-
sented here is in conformity with the earlier proposals made by W i n ~ t e i nin~explaining
~
similar processes in the nonchlorinated hydrocarbon systems. Considering the fact that
aldrin undergoes very slow skeletal rearrangement into the isodrin derived structure
(54) in a low yield (1.5%), the results cited here seem to indicate that removal of one
of the chlorine atoms at C-10 in the hexachloro compounds is possibly an essential
factor in accelerating protolytic rearrangement. Without this factor, the lifetime of a
carbonium ion such as (76) arising from the parent hydrocarbon would not be long
enough to allow rearrangement before neutralization of the charge by capture of the
solvent.
SCHEME 27
Interestingly, a similar compound (79) in which one of the C-10 hydrogens is re-
placed by t-butoxy group experiences only direct displacement of the t-butoxy group
by chloride ion t o afford (80) upon treatment with carbon tetrachloride and sulfuric
Volume I 189
acid.40 Ionization in this compound probably proceeds via a delocalized nonclassical

ion (81), which in turn is discharged stereospecifically by chloride ion as shown below.
VI. C H E M I C A L R E A C T I O N S O F D I E L D R I N , E N D R I N , A N D R E L A T E D
COMPOUNDS
The chemical conversions of dieldrin (6) and endrin (7) undoubtedly rest on the
reactivity of the epoxide ring present in these molecules, and apart from the normal
epoxide ring opening, a number of interesting transannular reactions and skeletal rear-
rangements have been observed.
As the cleavage of epoxide with aqueous acid normally proceeds with trans diaxial
opening, boiling of dieldrin with aqueous dioxane containing sulfuric acid gives the
transdiol (82).42Similarly dieldrin and hydrogen bromide in dry dioxane produce the
transbromohydrin (83)42(Scheme 28).
SCHEME 28
However, cis opening of the epoxide in dieldrin is also possible. Thus when dieldrin is
treated with an acetic acid-sulfuric acid mixture the cisdiacetate (84) is formed in ad-
dition to three other and treatment of this acetate with sodium methoxide
gives a cisdiol quantitatively (Scheme 29). This diol is found to be identical with (42),
which is obtainable from the oxidation of aldrin (Scheme 19).
SCHEME 29
A possible explanation can be made on the following grounds; the experimental con-
ditions limit the availability of acetate anions, thereby allowing the prior formation of
an ion pair such as (85), and then the attack of the anions would be such that it pro-
duces the cisdiacetate, or the rear approach of the bulky acetate ion would be sterically
hindered and their approach from the front side is obligatory.
In connection with a calorimetric method of estimating endrin, Skerret and BakeP4

reported that endrin was converted into the ketone (86) by boron trifluoride.
The same rearrangement was examined later by Cookson and C r u n d ~ e l l , and ~~.~~
the correct structure (57) was proposed for the product. This transformation involves
a transannular cyclization (87) + (88) and hydride shift in the resultant dipolar ion
(88) as shown in Scheme 30, which is closely related to Scheme 23. The transannular
hydride shift occurring in (88) as well as in (60) is not unusual in systems like these
where the steric requirements for transfer are easily fulfilled because of the proximity
of the reaction centers.
Shortly afterwards, Soloway and co-workers4' reported that the same rearrangement
of endrin could be effected under both thermal and acidic conditions. In this acid-
catalyzed rearrangement, the epoxide oxygen is first protonated, followed by transan-
nular cyclization (89) + (90) and a hydride shift (90) + (57) as has been already dis-
cussed in Scheme 30 and in Scheme 24. The discrete step is reproduced in Scheme 31.
SCHEME 30
SCHEME 31
During studies on the gas chromatographic behavior of endrin, Soloway and co-
noticed that endrin showed two peaks, neither of which was due to endrin
itself. The two compounds were then prepared by the thermal isomerization of endrin,
and one of them was identified as the well-known ketone (57). For the other com-
pound, a saturated aldehyde structure (91) was proposed mainly on the basis of the
mechanistic consideration of the isomerization. The same products (57) and (91) in the
ratio of 4:l were formed when endrin was irradiated under a UV lamp.47The thermal
isomerization of endrin to the aldehyde (91) presumably involves the same intermediate
(90) that produces (57), but instead of the hydride transfer as shown in Scheme 31,
the bond between C-6 and C-7 migrates to C-3 in order to neutralize the positive charge
at C-3 (Scheme 32). As a result, a carbonyl group is revealed at C-3 as an aldehyde.
Quite recently the correctness of the structure (91), with the exception of the confir-
guration of the carboxaldehyde which is in fact epimeric [dotted line in (91)], has been
confirmed by Bird and CO-workers.48
SCHEME 32
In an extensive study of the chromous chloride reduction of these insecticides,
C h a ~ found
,~ that reduction of endrin with chromous chloride gave rise to a pentach-
loro-pentacyclic ketone (92) in a high yield, acidic reaction conditions undoubtedly
transform endrin to the known ketone (57), which is subsequently dechlorinated to
(92).
Having repeated the acid-catalyzed rearrangement of endrin with neat sulfuric acid,
the present authors noticed the constant appearance of a minor product (6 to 8%)
besides (57) as the major Based on the 'H and 13C NMR spectra of the
minor product the structure (93) was assigned. The formation of (93) is rather unusual
and worthy of further comments since it is composed of C-C bond cleavage of an
epoxide accompanied by cycloaddition to a double bond. A plausible pathway for this
seemingly unusual transformation is presented in Scheme 33 together with the structure
of (93). The proposed structure has recently been confirmed by an X-ray crystal struc-
ture analysis.50b
SCHEME 33
Unlike the major pathway leading t o (57) (Scheme 31), the intermediate cation (90) is
probably partitioned into a different path in which the positive charge at C-3 is neu-
tralized by 1,3-bond shift of C-6,C-7 bond, assisted by attack of oxygen at C-7 rather
than by transannular hydride shift.
The ketone (57) itself was subjected to further investigations by several groups.
LiAlH, reduction of (57) gives an alcohol which is isomeric to (56) obtained from
hydrolysis of the acetate (54); therefore the structure (94) can be assigned.45 On the
other hand, when large excess of LiAlH, is used, the product is not the alcohol above
but a new pentacyclic alcohol (95).45
The formation of (95) may be rationalized as arising by electrophilic attack on oxygen

by aluminum hydride and transannular bonding between C-3 and C-6 using the elec-
tron pair made available by the loss of a proton. This mechanism is visualized in the
intermediate (96).
Following these observations, Winstein and co-workerss1 demonstrated that the

transformation of (57) t o (95) could be accomplished by treatment with alcoholic so-
dium hydroxide or simply by heating in pyridine. The same reaction by base was also
reported independently by FukunagaS2in an accompanying communication. An en-
tirely different mechanism to that of the LiAlH, reduction of (57) seems to be operat-
ing. According to the postulated mechanisms to which both groups agreed (Scheme
34),51.52the carbanion (98) is generated via a transition state such as (97) in which the
hydrogen atom at C-3 is abstracted by base with anchimeric assistance. The negative
charge at C-3 attacks carbonyl group to form the alkoxide anion (99). Protonation of
the anion (99) would give (95). By contrast, a similar ketone (100) was isomerized
almost quantitatively to ketone (101) upon treatment with potassium t-butoxide in t-
butyl alcohol (Scheme 35).52.53In this case an equivalent anion to (99) would be parti-
tioned between two pathways leading to (100) and (101), respectively, depending upon
their thermodynamic stability leading to (101) and (100) in the ratio of 94:6.
SCHEME 34 SCHEME 35
As a final example of chemical reactions of dieldrin, a Wagner-Meerwein type rear-

rangement (see Appendix) of dieldrin is presented. Boiling of dieldrin with acetic an-
hydride and sulfuric acid gives gem-diacetate (102) as a major product besides two
minor products (84) and (103) (Scheme 36).54The structure of (102) was characterized
on the basis of the detailed analysis of its 220 MHz NMR spectrum with decoupling
experiments at 100 MHz. The appearance of (102) and (103) would not be difficult to
imagine if one considered the intermediacy of a Wagner-Meerwein bridged cation. The
earlier observations made by Winstein and co-workers55clearly suggest the intrinsic
tendency of the aldrin skeleton to participate in the Wagner-Merwein rearrangement
if proper conditions are satisfied. Thus in the solvolysis of the bromobenzenesulfonate
(104), not only an unrearranged solvolysis product (105) but major amounts of rear-
ranged products (106) and (107) as the result of Wagner-Meerwein rearrangement form
in the reaction mixture. Apparently, the solvolysis accompanies the bridged cation
(108) upon which the formation of both (106) and (107) depends.
Volume I 193
SCHEME 36
The discrete steps are presumably as delineated in Scheme 37:(1) Wagner-Meerwein

shift [i.e., (108) (109)], (2) transannular bonding [i.e., (209) (1 10)], and (3) either
-+ +
bonding by the loss of a proton leading to the hydrocarbon (107) or discharge of the
ion by attack of OH- leading to (106). This reasoning also suggests that aldrin can
undergo Wagner-Meerwein rearrangement although reluctantly since it is clear that the
acetate (54) has its precursor in an ion like (108). Analogously, a mechanism shown in
Scheme 38 was proposed in order to accommodate our results, in particular the ap-
pearance of (102) and (103).
SCHEME 37
SCHEME 38
Thus the Wagner-Meerwein cation (11 l ) , is partitioned between two paths, and the
first path is the quenching of the positive charge by acetate ion, leading to (84) after
acetylation of the remaining hydroxy group. By way of the second route, a Wagner-
Meerwein 1,2-shift [(l 1 1 )+ (1 12)] followed by transannular bonding [(l 12) -+ (1 13)1,
leads from ion ( l l l ) t o the caged alcohol ( l 13). Loss of a proton and another transan-
nular bonding accompanied by trapping gives the acetate (103). Significantly, instead
of proton loss, hydride transfer occurs in (1 13) and probably triggers cleavage of the
C-8, C-9 bond with generation of a carbonyl group on C-9 and formation of a double
bond between C-7 and C-8. By this alternative pathway, the final product (102) can
be visualized as arising from the aldehyde (1 14).
VII. SUMMARY
The chemistry of the cyclodiene insecticides has been reviewed especially with respect
to their behavior in acidic or basic medium. As the foregoing examples manifest, tran-
sannular reactions and Wagner-Meerwein rearrangements are striking evidence that
these insecticides are provided with the intrinsic molecular compactness leading to in-
teresting and important chemical observations in the area of strained molecules. It is
gratifying to see that the chemistry of these species is continuing, yet much remains to
be explored in the future.
VIII. APPENDIX - THE WAGNER-MEERWEIN REARRANGEMENT
Throughout the review, there have been occasions in which molecules rearrange-
ments of the insecticides are explained in terms of a Wagner-Meerwein rearrangement.
As this term might be unfamiliar to some readers, it is felt that further description
would be helpful. A classical example, one of the most thoroughly studied cases, is
the camphene hydrochloride (1 15) to isobornyl chloride (1 16) rearrangement as shown
in Scheme 39.
SCHEME 39
Regardless of the detailed mechanistic discussions that have been set forth,s6 is has
been suggested that the cation (117) possessing a bridged structure is probably the
intermediate in this reaction and that chloride ion attacks the cation from the direction
indicated to lead to isobornyl chloride (1 16). The cation (1 17) is classified as an exam-
ple of a nonclassical ion, the existence of which is still inviting extensive argument
among organic chemists.=' In a formal sense, this rearrangement consists of the break-
ing of a C-C bond between C-l and C-6 of (1 15) and formation of a new bond between
C-2 and C-6; hence the overall process is equivalent to 1,2-migration of the C-1-C-6
bond. As this example shows, the term "Wagner-Meerwein rearrangement" refers to
the 1,2-migration of a ring member in a bridged bicyclic (or polycyclic) substance, and
the cases we have discussed in the previous sections all fall into the category of the
rearrangement.
IX. ACKNOWLEDGMENTS
We thank the National Research Council of Canada and Carleton University for
generous financial assistance.
Volume I 195
REFERENCES
1. Ungnade, H . E. and McBee, E. T., The chemistry of perchlorocyclopentenes and cyclopentadienes,

Chem. Rev., 58,249, 1958.
2. Riemschneider, R., The chemistry of the insecticides of the diene group, World Rev. Pest Control,
5,29, 1963.
3. Melnikov, N. N., Chemistry of pesticides, Residue. Rev., Volume 36, 1971.
4. Brooks, G. T., Chlorinated insecticides, Vol. I, Technology and application, Zweig, G., Ed., C.R.C.
Press Inc., Cleveland, Ohio, 1974.
5. Parlar, H. and Korte, F., Photoreactions of cyclodiene insecticides under simulated environmental
conditions, Chemosphere, 6,665, 1977.
6. Soloway, S. B., Ph.D. Thesis, University of Colorado, Boulder, 1955; quoted by Reference 4.
7. Eckroth, D. R., A method for manual generation of correct von Baeyer names for polycyclic hydro-
carbons, J. Org. Chem., 32, 3362, 1967.
8. IUPACnomenclature of Organic Chemistry, 3rd ed., Butterworth, London, 1971.
9. Benson, W. R., Nomenclature of dieldrin and related compounds, J . Assoc. Offic. Anal. Chem.,
52, 1109, 1969.
10. Wasserman, A., Diels-Alder Reactions, Elsevier, New York, 1965.
11. Onishenko, A. S., DieneSynthesis, Israel Program for Scientific Translations, Jerusalem, 1964.
12. Alder, K. and Stein, G., The course of the diene synthesis, Angew. Chern., 50, 510, 1937.
13. Woodward, R. B. and Hoffman, R., The Conservation of Orbital Symmetry, Verlag Chemie, GmbH,
Academic Press, Weinheim, 1970.
14. Straus, F., Kollek, L., and Heyn, W., Replacement of positive hydrogen by halogen, Chem. Ber.,
63B, 1868, 1930.
15. Prill, E. A., Diels-Alder syntheses with hexachlorocyclopentadiene, J. Am. Chem. Soc., 69.62, 1947.
16. Kleiman, M. and Goldman, A., l-Chlorochlordene, U.S. Patent 2,672,486; Chem. Abstracts, 48,
6642, 1954.
17. Lidov, R. E., Polycyclic Insecticides, U.S. Patent, 2,635,979; Chem. Abstracts,47, 6596, 1953.
18. Lidov, R. E. and Soloway, S. B., Polycyclic compounds and insecticidal compositions containing
the same, British Patent 692,547; Chem. Abstracts, 48, 324, 1954.
19. Arvey Corp., Synthesis of a compound related to aldrin and derivatives thereof, British Patent
714,688; Chem. Abstracts, 50,401, 1956.
20. Lidov, R. E., Polcyclic insecticidal compounds, U.S. Patent, 2,717,851; Chem. Abstracts, 50, 2914,
1956.
21. Chau, A. S. Y., Chromous Chloride reductions. 111. Identification of products obtained from pro-
longed contact of chlordene and heptachlor with chromous chloride solution, Bull. Environ. Contam.
Toxicol., 5,429, 1970.
22. Cochrane, W. P., Cyclodiene chemistry 11. Identification of the derivatives employed in the confir-
mation of heptachlor, heptachlor epoxide, cischlordane, and transchlordane residues, J. Assoc.
Off. Anal. Chern.,52, 1100, 1969.
23. Maher, J. R., Biemiek, D. and Korte, F., Beitrage zur okologischen Chemie liv Solvolyse von Hep-
tachlor und Dieldrin unter hohen Drucken, Chemosphere, 2, 31, 1973.
24. Metcalf, R. L., The Future for Insecticides; Needs and Prospects, vol. 6, Advances in Environ. Sci.
Technol., 1976, chap. 3.
25. Cochrane, W. P. and Chau, A. S. Y., Ring-opening isomerization of heptachlor epoxide with base,
Chem. Ind. (London), p. 1696, 1968.
26. Cochrane, W. P. and Forbes, H. A., lsomerization of l-ex@4,5,6,7,8,8-heptachloro-2,3-end@epoxy-
3a,4,7,7a-tetrahydro-4,7-methanoindane in base, Can. J. Chem.,49, 3569, 1971.
27. Davies, D. J., The addition of bromotrichloromethane and carbon tetrachloride to aldrin, J. Chem.
Soc., 3669, 1960.
28. Brace, N. O., Free radical addition of iodoperfluoroalkanes to bicyclic olefins, J. Org. Chem., 27,
3027, 1962.
29. Stedman, R. J., Swift, A. C., and Hoover, J. R. E., The reaction of isodrin with azides: the prepa-
ration of some new derivatives of the birdcage hydrocarbon, Tetrahedron Let., p. 2525, 1965.
30. McKinney, J. D., Keith, L. H., Alford, A., and Fletcher, C. E., The proton magnetic resonance
spectrum of some chlorinated polycyclodiene pesticide metabolites. Rapid assessment of stereochem-
istry, Can. J. Chem., 49, 1993, 1971.
31. McKinney, J. D. and H. M. Mehendale, Formation of polar metabolites from aldrin by pea and
bean root preparations, J. Agr. Food Chem., 21, 1079, 1973.
32. Bieniek, D., Moza, P. N., Klein, W., and Korte, F., Beitrage zur okologischen Chemie. XXIX.
Reduktive Dehalogenierung von chlorierten cyclischen Kohlenwasserstoffen, Tetrahedron Let., p.
4055, 1970.
33. Adams, C. H. M. and MacKenzie, K., Dehalogenation of isodrin and aldrin with alkoxide bases, J.
Chem. Soc. (C), p. 480,1969.
34. Bird, C. W., Cookson, R. C., and Crundwell, J., Cyclisations and rearrangements in the isodrin-
aldrin series, J. Chem. Soc. (C), p. 4809,1961.
35. Cookson, R. C. and Crundwell, E., Transannular reactions in the isodrin series, Chem. Ind. (Lon-
don), p. 703,1959.
36. Cookson, R. C. and Crundwell, E., Photochemical isomerization of isodrin, Chem. Ind. (London),
p. 1004,1958.
37. MacKenzie, K., The reaction of hexachlorolucyclo {2,2,l}heptadiene with potassium ethoxide, J .
Chem. Soc. (C), p. 457,1962.
38. Adams, C. H.M., MacKenzie, K., and Cawley, D. J., Sequential Wagner-Meerwein rearrangements
in the protolysis of monodechloraldrin and monodechloroisodrin into a unique unsaturated ketone,
Tetrahedron Let., p. 131 1, 1972; Sequential Wagner-Meerwein rearrangements of polychlorinated
complex cations derived from monodechloroisodrin and monodechloroaldrin, and the 'H nuclear
magnetic resonance spectra of polychloropolycyclic ketones and dechloroaldrin derivatives, J . Chem.
Soc., Perkin Trans. 11, p. 909, 1973.
39. Winstein, S. and de Vries, L., Neighbouring carbon and hydrogen. XXXIX. Complex rearrange-
ments of bridged ions. Rearrangement leading to the bird-cage hydrocarbon, J . Am. Chem. Soc.,
82,5363,1960.
40. Fletcher, A. V. and MacKenzie, K., Selective 'lithal' dehalogenation of polychlorotetracyclododeca-
dienyl alkylethers of the isodrin group and protolysis of the products, Tetrahedron Let., p. 1513,
1975.
41. Allred, E. L. and Hinshaw, J. C., n-electron participation in the endo, endedimethano-naphthad-
ienyl system, Tetrahedron Let., p. 1293,1968.
42. Parson, A. M. and Koore, D. J., Some reactions of dieldrin and the proton magnetic resonance
spectra of the products, J . Chem. Soc. (C), 2026,1966.
43. Chau, A. S. Y. and Cochrane, W. P., Cisopening of dieldrin oxirane ring, Chem. Ind. (London),
p. 1568,1970.
44. Skerrett, E. J. and Baker, E. A., A new colour reaction for dieldrin and endrin, Chem. Ind. (Lon-
don), p. 539, 1959.
45. Soloway, S. B., Damiana, A. H., Sims, J. W. Bluestone, H., and Lidov. R. E., Skeletal rearrange-
ments in reactions of isodrin and endrin, J . Am. Chem. Soc., 82,5377,1960.
46. Phillips. D.D.,Pollard, G. E., and Soloway, S. B., Thermal isomerization of endrin and its behav-
iour in gas chromatography, J . Agr. Food Chem., 10,217,1962.
47. Rosen. J. D.,Sutherland, D. J., and Lipton, G. R., Photochemical isomerization of dieldrin and
endrin and the effects on toxicology, Bull. Environ. Contam. Toxicol., l, 133, 1966.
48. Bird, C. W., Khan, R., and Richardson, A. C., Structure of endrin aldehyde, Chem. lnd. (London),
p. 231,1978.
49. Chau, A. S. Y., Chromous chloride reductions. IV. Reaction of endrin with chromous chloride so-
lution, Bull. Environ. Contam. Toxicol.,S, 435,1971.
50a. ApSimon, J. W., Yamasaki, K., Fmchier, A., and Chau, A. S. Y., Carbon-carbon bond cleavage in
an epoxide. A minor product from the acid treatment of endrin, Tetrahedron Let., 3677,1977.
50b. ApSimon, J. W., Huber, C., and Yamasaki, K., unpublished observations.
51. Carter, P., Howe, R., and Winstein, S., Preparation and solvolytic behaviour of a bridghead birdcage
alcohol, J . Am. Chem. Soc., 87,914,1975.
52. Fukunaga, T., Isomerization via transannular enolate anion, J . Am. Chem. Soc., 87,916,1%5.
53. Howe, R. and Winstein, S., Homoenolization-homoketonization of a half-cage ketone, J . Am.
Chem. Soc., 87,915,1965.
54. ApSimon, J. W., Buccini, J. A., and Chau, A. S. Y., The acid-catalyzed Wagner-Meerwein rear-
rangement of dieldrin, Tetrahedron Let., p. 539,1974.
55. Bruck, P., Thompson, D., and Winstein, S., Dechlorination of isodrin and related compounds,
Chem. Ind. (London), p. 405, 1960.
56. Berson, J. A., Carbonium ion rearrangements in bridged bicyclic systems, in Molecular Rearrange-
ments, Part One, deMayo, P., Ed., John Wiley & Sons, New York, 1963.11 1.
57. Brown, H. C. and Schleyer, P. V. R., The Nonclassical Ion Problem, Plenum Press. New York,
1977.
INDEX
BFJMeOH, 132, 144

BHC, see Benzene hexachloride
Abiotic reactions to pesticides, 2 Biological degradation, 13
Ac,0,99 Biological reactions, 2-4
Ac,O/HISo,, l l 5 Biotransformation, 9
Accumulation, 11-12 BITB, see bis-Isopropoxytetrachlorobenzene
mechanisms of, l 3 Boron trichloride, 99
natural system, l I Boron trifluoride, 102
Bromination, 123, 142-145
I-Acetoxy-3-chlorochlordene, 108 Bromoacetate, 104
Acetylation, 113, 115, 116, 118, 131 transBromoacetate, 104
Acetylcholinesterase, 3-4 Bromohydrin, 104
Acid-catalyzed isomerization, 99, 102 cisBromohydrin, 104
Acid cleanup, 35 transBromohydrin of dieldrin, 104
Adsorbents, 73 BSTFA, I40
Adsorption, 9 t-BuOK, 96,97
Adsorption column elution pattern, 5 1 t-BuOK/ t-butanol (BuOH), 113, 114
AFID, see Alkali flame ionization detector t-Butanol (t-BuOH), 96, 97, 106
AgOAc/HOAc, 108 t-Butyl hypochlorite (t-BuOCI), 106
Alder's rule, 178 t-Butyl hypochlorite (t-BuOCl)/HOAc, 106, 109
Aldicarb, 138, 149
Aldrin, 86, 105, 107-109, 121, 125, 128, 158,
176, 180, 183-185.187, 188, 193
Alkali flame ionization detector (AFID), 46, 136,
140, 144, 151, 152, 154 Carbamates, 2.4, 28, 138, 139, 141, 144
Alkaline cleanup, 36 solubility of, 7
Alkaline dehydrochlorination, 95 Carbaryl, l49
Alkaline precolumn, 113, 125, 126 Carbophenothion, l36
Alkoxyl ethers, 118 Carrier gas, 42
Alkylation, 137, 139, 140, 151-155 CCD, see Coulson conductivity detector
Allylic chlorine, 108 CD-GLC, 87,93, 158
Allylic reductive dechlorination, 108 Chemical cleanup, 35
Alumina, 33, 114 Chemical confirmatory tests, 55
Aluminum chloride, 123 Chemical derivatization, 85, 87
Ametryne, 149 CH,I, 155
Aminofenitrothion, 141 CH,/K,CO,, 132
Amino parathion, 141 Chloracetylation, 145
Analytical methodology, 59 Chlordane, 86, 97, 108, 111, 113, 114, 175, 179
Analytical reference standards, 58 technical, 108, 114
Antimony pentachloride, 109, 123 cischlordane, 86, 89, 97,99, 108, 113, 114, 183
Aquatic ecosystems, 6 transchlordane, 86.97, 108, 113, 114, 176, 183
Artifacts, 71, 86 Chlordene, 108, 109, 158
Asym-triazine, see 1.2.4-Triazine Chlorination, 123, 158
Atmosphere, 6 Chlorine, 108
Atmospheric deposition, 10 transchloroacetate, 104
Atmospheric fallout, 6 Chloroacetic anhydride, 107
Atratone, 150, 151, 155 2-Chlorochlordane, 86, 113
Atrazine, 148-154 3-Chlorochlordene, l l 3
Azodrin, 138, 139 2-Chloroethylation, 144
2-Chloroethylesters, 93
mChloroperbenzoic acid, 107
2-Chloro-striazine, 149-152
Chloroxuron, l47
Base-acid partitioning, 36 Chromatography, see specific types
Base-catalyzed intramolecular reaction, 115 Chromic acid, 107
BCl,/2-chloroethanol, 105 Chromic oxide, 109
Benzene hexachloride (BHC), 123, 126, 127 Chromogenic solution, 85
a-Benzene hexachloride (BHC), 128, 129 Chromous chloride, 108, 109, 141, 158
BFJdiethyl ether, 103 dechlorination by, 97
Chromous chloride reductive dechlorination, 120 Degradation, 5, 7-1 1, 16, 28

Cleanup, 3 1 DDT, 13
acid, 35 factors in, l 5
alkaline, 36 mechanisms of, l 3
chemical, 35 sediment influence on biological, 13
column, 32 Dehydrochlorination, 3,97, 114, 125-127
defined, 31 alkaline, 95
CO-distillation, 37 of DDT, 95,97, 119, 130, 136
CO-extractives, 70 Demeton-SMe, 138
Column chromatography, 187 Deposition, 6, 7
Column cleanup, 32 atmospheric, 10
Column efficiency, 42 Derivatization, 89-93
Column material, 39 chemical, 85, 87
Column packings, 40 flash-heater, 136
Concentrated stock solutions, 48 G C technique, 115
Concentration-time profiles, 16 GLC-chemical, 144
Confirmation, see Confirmatory techniques in-block GC, 136
Confirmatory techniques, 76, 84, 85 inlet block, 136
Coulson conductivity detector (CCD), 48, 140, on-column, 136
144 organochlorines, 87
Counter, 139 photochemical, 87
sulfoxides of, 138 SM, 101, 112, 143
CrCI,, 129, 130 Detection, 136
CrC1,-ethylenediamine complex, 121 Detectors
CrCI, reaction, 102 alkali flame ionization, 46, 136
CrC1, reduction, 137 Coulson conductivity, 48, 140, 144
CrO,, 99 electrolytic conductivity, 48
Crufomnate, 137 electron-capture, 44
Cyanox, 136 flame photometric, 47, 144
Cyclodiene synthesis, 177 gas chromatographic, 44
Cyclopentadiene, 3 Hall electrolytic conductivity, 48
microcoulometric, 48
nitrogen-phosphorus, 46
non-specific, 137
specific, 53
2.4-D, 143 thermionic, 144
Dasanit, 138 Diacetate, 115, 116, 118
2.4-DB, 143 Dialkyl phosphate, 131
DBU (1,5-diazobicycle (5.4.0.) un-dec-5-ene), 97 0,ODialkyl phosphorothionate, 131
transDCS ( transdichlorostilbene), 97-98 Diazinon, 140
0 , ~ ' - D D D96
, Diazomethane, 129, 132
p,p'DDD, 96-98, 106 cisDibl-on~ide,105
DDE, 96 transDibromide, 105, 106
dehydrochlorination of DDT to, 97 transDichloride, l05
o,p'-DDE, 86,97 transDichloroaldrin, 106
p,p'-DDE, 86,95, 99, 128 Dichlorobenzene, 145
DDMU, 97.98 2,4-Dichloro-6-(-0chloroanilino)-striazine, see
DDNU, 98 Dyrene
DDT, 86, 89, 94,95, 97,98, 113, 114 p,p-Dichlorobenzophenone, 99
degradation of, 13 1,2-Dichlorochlordene, 114
dehydrochlorination of, 95, 97, 119, 130, 136 Dichlorofenthion, 136
olefins of, 95 Di-(pchlorophenyl)-acetic acid, 96
o,p'-DDT, 96.97, 128 Dieldrin, 86, 100, 101, 103, 107, 108, 119, 121,
p,p'-DDT, 95-97, 106, 107 158, 177, 180, 189, 192
Dealkylation of triazines, 5 transbromoacetate of, 104
Decachlorobiphenyl, 120 transbromohydrin of, 104
Dechlorination, 121, 124 Diels-Alder adduct, 179
allylic reductive, 108 Diels-Alder reaction, 177-178, 180, 181
by chromous chloride, 97 Diethyl phosphate, 131
by nickel boride, 97 Diethyl phosphorothionate, 131
by sodium naphthalenide, 97 2.8-Dihydro, 121, 122
reductive, 3, 102, 109, 120 5,10-Dihydro derivatives, 122
Volume 1 199
Di-methoxy derivatives of Dyrene, 15 1

Dimethyl phosphate, 13 1, 132
Dimethyl phosphorothionate, 13 1 Gas chromatography (GC), 84, 102, 103,125,
Dimethyl sulfate, 132 128, 130,143,148,157-159
2.4-Dinitrofluorobenzene, 152 derivatization, 89
Diol, 1 1 5 detectors for, 44
Disilyl ether, 116 reaction, 113
Diuron, 146, 147 Gas chromatography (GC)/mass spectrometry
DNFB, 152,153 (MS), 84.87, 155, 156
DNT/DNP, 145 Gas-liquid chromatography (GLC), 38,39, 50,
Drinking water, 8 72,84,88,102, 109,114, 119-121, 123,
Dyfonate, 136 129-132, 136,140, 142, 144-149, 153,
Dyrene, 149, 150 154, 158, 159
di-methoxy derivatives of, 151 maintenance of column, 44
mono-methoxy derivatives of, 151 multi-column, 85,86
pre-column, 136
GC, see Gas chromatography
Gel-permeation chromatography (GPC), 37
Geographical differences
ECD, see Electron-capture detector in fallout, 6
Efficiency of columns, 42 in herbicide concentration levels, 8
Electrolytic conductivity detectors, 48, 140, 144 Glass rod TLC, 77
Electron-capture detector (ECD), 44,50,72,88, GLC, see Gas-liquid chromatography
98,107,109, 110, 113, 118,121,123, 137, GPC, see Gel-permeation chromatography
140, 142, 144, 151, 156
Elution pattern, 51
Emulsion, 69
Endosulfan, 116-1 18
a-Endosulfan, 86, 112, 115, 119
Hall electrolytic conductivity detector (HECD),
P-Endosulfan, 112,1 1 5, 119
48
Endosulfan acetate, 116
HBr, 104
Endosulfan ether, 118
HBr,Ac,O, 104, 113
Endrin, 99, 101, 102, 108, 114, 119, 180,
HBr/HOAc, 104
189-191
HCB, see Hexachlorobenzene
Endrin ketone, 99
HCH, see Hexachlorocyclohexane
EPN, 141
HCI, 99
Epoxidation, 3, 109
HCI/Ac,O, 105, 113, 118
Ethephon, l32
HCl/MeOH, 132
Ether, 115, 116
HCIO,, 103
Ethylene glycol, 129
HECD, see Hall electrolytic conductivity detector
Etrimfos, 137
Heptachlor, 86,97.99,102, 108,109, 112-1 14,
Evaporation, 61.64
121, 128, 129, 158, 175, 176, 180-182
Expoxidation, 158
Extraction, 30 Heptachlor epoxide, 97.99, 108, 113, 114, 119
Extraction p-value, 53 Heptachlorobiphenyl, 122
Herbicidal acids, 28,93
Herbicide concentration levels. 8
Herbicide solubility, 7
Hexachlorobenzene (HCB), 123, 127, 130
Fallout, 6 Hexachlorocyclohexane (HCH), 123
Fenitro oxon, 141 Hexachlorocyclopentadiene, 179
Fenitrothion, 136, 141 High-performance TLC (HPTLC), 78
Fenuron, 146,148,151 High-pressure liquid chromatography (HPLC),
Films, 7 53, 145,146,159
Flame photometric detection, 136 Homologous series, 142
Flame photometric detector (FPD), 47, 140, 144 Hot plate TLC, 77
Flash-heater derivatization, 136 HPLC, see High-pressure liquid chromatography
Flenuron, 147 HPLS-UV, 146
Florisil, 33 HPTLC, see High-performance TLC
Fluometuron, 147 HZS0,/Ac20, 101
Formaldehyde, 136 H,SO,/propanol, 143
FPD, see Flame photometric detector Hydrobromic acid, 107
Hydrochloric acid, 102 Methoxylation. 152-154

Hydrolysis, 2, 3, 4 2-Methoxy-striazines, 150
Hydroxyatrazine, 151 Methylation, 131, 144, 148, 151, 153-155
l-Hydroxy-3-chlorchlordene, 1 13 "inlet block," 132
1-Hydroxychlordene, 93, 108, 112-114 NMethylation, 146, 149
Methyl fluorosulfate, 132
Methyl parathion, 136, 138
Metoprotryne, 149
MFO, see Mixed-function oxidases
In-block G C derivatization, 136 Micro-calorimetric determination, 94
Indene, 175 Microcoulometric detector (MCD), 48
Infrared spectroscopy (IR), 54, 85 Microlayer, 15
"lnlet block" derivatization, 136 Mirex, 120-123, 141, 158
"lnlet block" methylation, 132 Mixed-function oxidases (MFO), 3-4
Interferences, 71 Monitoring, 2, 8, 9, 17-18, 27
Intermediate concentration stock solutions, 49 in prairies, 8
Iodination, 145 of sediments, 10
IPB, see lsopropoxypentachlorobenzene Monohydro derivatives, 122
IR, see Infrared spectroscopy 8-Monohydro derivatives, 122
Isodrin, 176, 180, 183-188 Mono-methoxy derivatives of Dyrene, 151
Isomerization, 99, 102 Monoperpthalic acid, 107
Isopropoxypentachlorobenzene (IPB), 128, 129 Monopropoxylpentachlorobenzene
bis-lsopropoxytetrachlorobenzene(BITB), 128, (pentachlorophenyl propyl ether), 128
129 Monuron, 147
Movement of pesticides, 5-1 l
MS, see Mass spectroscopy
Multi-column gas-liquid chromatography (GLC),
85,86
Multi-dimensional TLC, 77
Kepone, 120, 122, 123, 158
KOH/ROH. 118
NaH/CH,/DMSO,, 139, 140, 147, 148, 151, 153,

154
LAH, 118 NaOMe/MeOH, 113, 125, 126
Landrin, l38 Natural system accumulation, 11
Lindane, 109, 123, 125, 126, 129 Nickel boride, 127
Linuron, 146, 147 dechlorination by, 97
Liquid-liquid partitioning, 3 1 Nitration, 120, 122
Liquid-solid chromatography, 32 Nitrogen-phosphorus detector (N-PD), 46
Lithium aluminurn hydride (LAH), 115 NMR, see Nuclear magnetic resonance
Nomenclature, 174
Nonachlor, 97, 114
Non-specific detector, 137
N-PD, see Nitrogen-phosphorus detector
Nuclear magnetic resonance (NMR), 54, 85, 191,
Marine sediments, 11 192
Marine waters, 8
Mass spectroscopy (MS), 54, 84, 85, 87, 155, 156
MCD, see Microcoulometric detector
MCP, 144 0.c.s. see Organochlorines
MCPA, 143 Olefins, 94.95
MCPB, 144 On-column alkylation, 139
MCPP, see Mecoprop On-column derivatization, 136
Mecoprop (MCPP), 143, 144 On-column transesterification, 132
Meobal, 149 0.p.s. see Organophosphates
Mesurol, 138, 148 Organochlorines (o.c.s), 2-3,28,93, 130, 141
Methanol, 125 derivatization techniques for, 87
Methiocarb, 149 solubility of, 7
Methomyl, 149 Organophosphates (o.p.s), 2, 28, 131, 132, 137,
Methoxychlor, 94, 96, 114 138
acetylcholinesterase biological reactions of,
3-4
photochemical reaction of, 3-4 Quality control, 55
solubility of, 7
0V-225 column, 86
Oxidases, 3-4
Oxidation, 137, 142
Oxydemeton methyl, 138, 139
Reaction gas chromatography, 89, 113
Reactions to pesticides, 2
Reductive dechlorination, 3, 102, 109, 120
allylic, 108
Reference materials, 59
Paraoxon, l41 Reference standards, 58
Parathion, 136, 141 Resins, 3 1
Partition coefficient, 86 Resolution, 42
Partitioning Resuspension, 7
base-acid, 36 Reversed-phase TLC (RP-TLC), 77
liquid-liquid, 31 Ronnel, 136
PCB, 106, 112, 116, 120, 122, 130 Rotary evaporator, 64
PCl,, 154 RP-TLC, see Reversed-phase TLC
Pentachlorophenol, 127, 129, 130 Rufomate, 136
Pentachlorophenyl propyl ether
(monopropoxylpentachlorobenzene), l28
Pentafluorobenzoate, 137
Pentafluorobenzoyl chloride, 137 Samples
Pentafluorobenzylation, 145 handling of, 29
Pentafluorobenzyl (PFB) ether, 136 preparation of, 30,61
Peracetic acid, 107 preservation of, 29
Perbenzoic acid, 107 storage of, 29
Perchlorination, 120, 122, 123, 127 Sediments, 9, 15
Performic acid, l07 adsorption to, 9
Persistence, 13 influence of o n biological degradation, 13
Perthane, 94-96 marine, 11
Pest, defined, 2 monitoring of, 10
Phenoxyalkanoic acid, 2.4, 142 transport of, 10
NPhenyl carbamate, 146 Selenium dioxide, 116
l-Phenylchlordene, 110 Sencor, 149, 151, 153, 154
Phorate, l36 Silica gel, 33
sulfoxide of, 138, 139 Silver carbonate, 108
Phosphoramidothioates, 139 Silylation, 113, 115, 140, 145, 151, 153, 154
Phosphorothionate pesticide, 3 Simazine, 150, 153, 154
Phosphorous pentachloride, 122, 123 Simetone, l50
Photochemical confirmatory tests, 55 Simulation, 15-17
Photochemical reactions, 2-4,87 SM, see Solid matrix
Photolysis, 98, 108, 119, 122 Sodium methoxide, 125
Photometric detection, 136 Sodium naphthalenide, 97
Photomirex, 121 Solid matrix (SM) derivatization, 89-93, 101,
Photosensitizing, 3 110, 112-116, 119, 143
Polyurethane foams, 3 1 Solid support, 39
Prairies, 8 Solubility, 7, 9
Precolumn technique, 125, 126, 136 Solvent purity, 30, 68
Preparation of samples, 61 Solvent replacement, 64
Preservation of samples, 29 Specific detectors, 53
Prometone, 150, 151, 154, 155 Spectrochromatogram, 85
Prometryne, 149, 151, 153 Spectroscopy, 54, 85
Propazine, 150, 153, 154 Standard reference materials (SRM), 59
Properties of pesticides, 2 Standards
Propham, 146, 147 analytical reference, 58
P-value, 53, 85, 86 of stock solutions, 49
Pyridine, 130 Standard solutions, 48
Pyrolysis, 98 Stationary phase, 40
Steady state, 16 Triazines, 2, 141, 149

Stock solutions, 48.49 dealkylation of, 5
Storage of samples, 29 substitution of, 5
Substituted ureas, 5 1,2,3-Trichlorobenzene, 125, 126
Substitution of triazines, 5 1.2.4-Trichlorobenzene, 124, 125
Sulfonylation, 145 1.3.5-Trichlorobenzene, 125, 126
Sulfoxides of phorate and Counter, 138 Trifluoracetylation, 139
Sulfur, 86 Trifluoroacetic anhydride, 138
removal of, 69 Trifluoroacetylation, 137, 138, 148, 149
Sulfuric acid, 99 Trimethylanilinium hydroxide (TMAH), 132
Surecide, 141 Trimethyl phosphate, 132
Surface films, 7 Two-dimensional TLC, 77
Surface microlayer, 15
Suspended materials, 7, 15
Sweep CO-distillation,37
Synthesis of cyclodiene, 177
Ultraviolet (UV) radiation, 137
Ultraviolet (UV) spectroscopy, 54
Urea, 2,28, 138, 139, 141, 144, 146
substituted, 5
TCDDs, 4 UV, see Ultraviolet
Tcrbufos, 139
Thcrrnionic detector, 144
Thin-layer chromatography (TLC), 34, 52,72,85,
124, 145, 157, 159
coating of plates in, 73 V-triazine, see 1,2,3-Triazine
confirmation of identity and, 76
glass rod, 77
high-performance, 78
hot plate, 77
multi-dimensional, 77
Wagner-Meerwein rearrangement, 188, 192, 193
reversed-phase, 77
defined, 194
two-dimensional, 77
Water, 7
Thin-layer chromatography (TLC)-GC, 156
drinking, 8
Thin-layer chromatography (TLC)-GLC, 75
marine, 8
2-Thiomethyl-striazines, 149
physical-chemical behavior of pesticides in, 7
Thiono-thiolo isomerization, 138
removal of, 66
Thiophosphorylation, 145
solubility in, 7
Thymol, 137
TLC, sec Thin-layer chromatography
TMAH, see Trimethylanilinium hydroxide, 132
Toxicology, l 7
2.4.5-TP, 143
Transesterification. 93, 142, 144 XAD resins, 3 1
on-column, 132
Transport of pesticides, 5-1 1
1,2,3-Triazine (v-triazine), 149
1,2,4-Triazine (asym-triazine), 149
~ T r i a z i n e28
,

Analysis of Pesticides in Water. Volume I, Significance, Principles, Techniques, and Chemistry of Pesticides by Afghan, B. K. Chau, Alfred S. Y. Robinson, James W

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Analysis of Pesticides in Water. Volume I, Significance, Principles, Techniques, and Chemistry of Pesticides by Afghan, B. K. Chau, Alfred S. Y. Robinson, James W

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CRC SERIES IN ANALYSIS FOR

ANALYSIS OF PESTICIDES IN WATER

Alfred S. Y. Chau, Senior Editor

CRC Press is an imprint of the

Reissued 2018 by CRC Press

© 1982 by CRC Press, Inc.

No claim to original U.S. Government works

Library of Congress Cataloging in Publication Data

Main entry under title:

Significance, principles, techniques, and chemistry of

(Analysis of pesticides in water; v. 1) (CRC series

A Library of Congress record exists under LC control number: 81010064

ISBN 13: 978-1-315-89056-2 (hbk)

The assessment of the environmental impact of man's endeavors must be made as

J. W. Robinson is Professor of Chemistry and Chairman of the Analytical Division

Alfred S. Y. Chau is Head, Quality Assurance Methods Section of the Analytical

B. K. Afghan is research scientist in environmental analytical chemistry in the Ana-

Professor John W. ApSimon, P h D . Derek Muir, Ph.D.

Environmental Impact and Significance of Pesticides

ENVIRONMENTAL IMPACT AND SIGNIFICANCE OF PESTICIDES

W.M. J . Strachan. W.A. Glooschenko. and R .J . Maguire

I1. Pesticide Types and Properties ......................................... 2

111. Transport and Movement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

VIII . Monitoring ........................................................ 17

11. PESTICIDE TYPES AND PROPERTIES

hexachlorocyclo- hexachloro- diphenyltrichloro- chlorinated cyclopenta-

phosphorothionates phosphorothiolates phosphorodithioates

By far the most common is the phosphorothionate pesticide. Completely oxygenated

D. Phenoxyalkanoic Acid Derivatives

111. TRANSPORT AND MOVEMENT

Pesticides enter the water from various sources. E d w a r d ~reports

FIGURE 1. Dynamic movement of pesticides in the aquatic en-

a southern Swedish lake was made by S ~ d e r g r e nwho

1. Organochlorines - 0.0012 mg/l for DDT to 7 mg/l for lindane

It is apparent that a pesticide may be present in water in a dissolved form or as a

2,2- DICHLOROETHANOL 2,2- DICHLOROETHANE 2 - CHLOROETHYLENE

FIGURE 2. Major microbial degradation pathways of DDT (R=4-chlorophenyl)

The use of simulation models to describe chemical behavior in aquatic environments

BASIC PRINCIPLES AND PRACTICES ON THE ANALYSIS OF

Alfred S . Y . Chau and Hing-Biu Lee

111. Preparation of Standard Solutions ..................................... 48

IV . Confirmation of Pesticide Identity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

V. Some Aspects of Quality Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .55

V11. Thin-Layer Chromatography (TLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

11. GENERAL SEQUENCE FOR PESTICIDE RESIDUE ANALYSIS

A. Sampling, Sample Handling, Storage, and Preservation

2. Selection of a Solvent System

Solvent-solvent partitioning is a cleanup procedure used to remove significant quan-

2. Liquid-Solid Chromatography (Column Cleanup)

3. Thin-Layer Chromatography (TLC)

Note: Alumina has been ap-

6. Gel-Permeation Chromatography (GPC)

E. Gas-Liquid Chromatography (GLC)

1. Be inert to prevent catalytic decomposition of labile compounds, adsorption, and

Flux-calcined diatomaceous earth materials such as Anakrom, Chromosorb W HP,

d . Preparation of Column Packings

SE-30 Methyl silicone, gum low

e. Packing and Conditioning o f a New Column

1. If the column is intended to be a certain length, it should be packed to that length

g. Column Efficiencyand Resolution

h. Maintenance o f a GLC Column

3. Gas Chromatographic Detectors

a. Electron-Capture Detector (ECD)