Pesticide Residues in Food: Technologies

for Detection

October 1988

NTIS order #PB89-136444
Recommended Citation:

U.S. Congress, Office of Technology Assessment, Pesticide Residues in Food: Technologies for Detection, OTA-F-398 (Washington, DC: U.S. Government Printing Office,

October 1988).

Library of Congress Catalog Card Number 88-600566

For sale by the Superintendent of Documents

U.S. Government Printing Office, Washington, DC 20402-9325

(order form can be found in the back of this report)
Foreword

Pesticides are an integral part of agriculture today, but their use can lead to

residues in agricultural products. Because of their potential adverse human health

effects, the Federal Government sets limits on allowable levels of pesticide residues

in food and animal feed and monitors these products to enforce those levels.

Federal monitoring and enforcement action is dependent on technical capability to detect pesticides. A major concern is that Federal regulatory agencies cannot

practically monitor food for all pesticides of health concern. OTA was asked to

assess whether existing and emerging technologies could improve Federal monitoring of pesticide residues in food. In addition, OTA examined the Federal research programs dedicated to improving Federal analytical capabilities for the

detection of pesticides in food.

This study was requested by the House Committee on Energy and Commerce

Subcommittee on Oversight and Investigations; the House Committee on Agriculture; and its Subcommittee on Domestic Marketing, Consumer Relations, and Nutrition. The Senate Committee on Agriculture, Nutrition, and Forestry and the House

Agriculture Subcommittee on Department Operations, Research, and Foreign Agriculture endorsed the request.

OTA appreciates the valuable assistance of the study’s workshop participants

and observers, authors of commissioned technical papers, and the many other individuals from the public and private sectors who provided information throughout

the course of this assessment and reviewed a draft of the report. As with all OTA

studies, the content of this report is the sole responsibility of OTA.

. . .

I l l
— ——

OTA Workshop Participants:

Technologies To

Henry Conacher

Food Research Division

Health and Welfare Canada

Ottawa, Canada

John Cowell

Environmental Sciences Department

Monsanto Co.

St. Louis, MO

William Cusick

Chemistry Laboratory Services

Department of Food and Agriculture

Sacramento, CA

Richard Ellis

Chemistry Division

Food Safety and Inspection Service

U.S. Department of Agriculture

Washington, DC

Silvia Canseco Gonzalez

Plant and Animal Health Office

Agriculture Department

Cozoacan, Mexico City

Kenneth Hill

Environmental Chemistry Laboratory

Detect Pesticide Residues in Food,

Mar. 14-16, 1988

Ralph Mumma

Pesticide Research Laboratory

Pennsylvania State University

University Park, PA

W. Harvey Newsome

Pesticide Section

Food Research Division

Health and Welfare Canada

Ottawa, Canada

Cynthia Peck

Analytical and Environmental Chemistry

Dow Chemical Co./USA

Midland, MI

Jack Plimmer

Environmental Chemistry Laboratory

Agricultural Research Service

U.S. Department of Agriculture

Beltsville, MD

Leon Sawyer

Center for Food Safety and Applied Nutrition

Food and Drug Administration

U.S. Department of Health and Human Services

Washington, DC

Agricultural Research Service -

Jim Seiber

U.S. Department of Agriculture Department of Environmental Toxicology

Beltsville, MD University of California

Kenneth Hunter

Westinghouse Bio-Analytic Systems Corp.

Rockville, MD

Lyle Johnson

Analytical Bio-Chemistry Laboratories, Inc.

Columbia, MO

Bruce Kropscott

Analytical and Environmental Chemistry

Dow Chemical Co./USA

Midland, MI

Harry Lento

Corporate Analytical Chemistry

Campbell Soup Co.

Camden, NJ

Julius Menn

Plant Science Institute

Agricultural Research Service

U.S. Department of Agriculture

Beltsville, MD

Davis, CA

Sarah Taylor

Congressional Research Service

The Library of Congress

Washington, DC

Charles Trichilo

Residue Chemistry Branch

Office of Pesticides and Toxic Substances

Environmental Protection Agency

Washington, DC

NOTE: OTA appreciates and is grateful for the valuable assistance and thoughtful critiques provided by the workshop

participants. The workshop participants do not, however, necessarily approve, disapprove, or endorse this

report. OTA assumes full responsibility for the report and the accuracy of its contents.

iv
OTA Project Staff

Pesticide Residues in Foods: Technologies and Methodologies

for Detection

Roger C. Herdman, Assistant Director, OTA

Health and Life Sciences Division

Walter E. Parham, Food and Renewable Resources Program Manager

Susan Shen, Project Director

Contractors

H. Anson Moye, University of Florida

Allen Ruby, In-House Contractor

Lisa Olson, Editor

Adminisrative ● aff

Ellis Lewis, Administrative Assistant

Nellie Hammond, Secretary

Carolyn Swann, Secretary
Abbreviations and Acronyms

ADI

AFID

AOAC

CCPR

CDFA

CES

CFSAN

DDT

ECD

EDB

ELISA

EPA

FDA

FFDCA

FIA

FIFRA

FMIA

FOI

FPD

FR

FSIS

FTE

GC

GLC

GPC

HECD

HPLC

IR

ITD

I UPAC

—Acceptable Daily Intake

—alkali flame ionization detector

—Association of Official Analytical

Chemists

—Codex Alimentarius Committee on

Pesticide Residues

–California Department of Food and

Agriculture

—compound evaluation system

–Center for Food Safety and Applied

Nutrition (of FDA)

—dichlorodiphenyl trichloroethane

—electron capture detector

—ethylene dibromide

—enzyme-linked immunosorbent assay

—Environmental Protection Agency

—Food and Drug Administration

—Federal Food, Drug, and Cosmetic

Act

—Federal Insecticide Act

—Federal Insecticide, Fungicide, and

Rodenticide Act

—Federal Meat Inspection Act

—Freedom of Information

—flame photometric detector

—Federal Register

—Food Safety and Inspection Service

—full-time equivalents

–gas chromatography

–gas liquid chromatography

—gel permeation chromatography

—Hall microelectrolytic conductivity

detector

—high performance liquid chromatography

—infrared (detector)

—ion trap detector

—International Union of Pure and Applied Chemistry

LC

LIMS

LOQ

LUO

MOG

MOU

MRP

MRL

MRM

MS

MSD

NBS

NOEL

NPD

NRP

NTIS

OMA

PAM

Pc

PCB

PICRC

RRT

SF

SFC

SFE

SI

SIM

SPE

SRM

TDRC

TDS

TLC

UAR

–liquid chromatography

—Laboratory Information Management

Systems

—limits of quantitation

—laboratory unit operations

—Mills, Onley, and Gaither

—memorandum of understanding

—multiresidue procedure

—maximum residue limits

—multiresidue method

—mass spectrometry

—mass selective detector

—National Bureau of Standards

—No Observable Effect Level

—nitrogen-phosphorus detector

—National Residue Program

—National Technical Information Service

—Official Methods of Analysis

—Pesticide Analytical Manual

—paper chromatography

—polychlorinated biphenyls

—Pesticide and Industrial Chemicals

Research Center (of FDA)

—relative retention time

—supercritical fluid

—supercritical fluid chromatography

—supercritical fluid extraction

—Surveillance Index

—single ion monitoring

—solid phase extraction

—single residue method

–Total Diet Research Center (of FDA)

–Total Diet Study

—thin layer chromatography

—unidentified analytical response

USDA/ARS—United States Department of Agriculture/Agricultural Research Service

UV/VIS —ultraviolet-visible (light detector)
Contents

Page

Abbreviations and Acronyms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

Chapter 1:

Chapter 2:

Chapter 3:

Chapter 4:

Chapter 5:

Chapter 6:

Chapter 7:

Chapter 8:

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Federal Pesticide Residues in Food Monitoring Programs . . . . . . . . . . 7

Contemporary Analytical Techniques for Pesticide Residues

in Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Immunoassay: An Emerging Technology. . . . . . . . . . . . . . . . . . . . . . . . . 37

Automation in Today’s Pesticide Laboratory . . . . . . . . . . . . . . . . . . . . . 49

Pesticide Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Federal Methods Development Programs for Detecting Pesticides

in Food . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

Summary and Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

Appendix A: OTA Workshop Participants and Observers . ...................109

Appendix B: OTA Workshop papers . . . . . . . . . . . . . . . . . . . . . . . . . . ............112

Appendix C: Glossary of Terms . . . . . . . . . . . . . . . . . . . . . . . ...................230

vii
Chapter 1

Introduction
—

Contents

Introduction . . . . . . . . . . . . . . . . . . .

Chapter 1 References . . . . . . . . . . .

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Chapter I

Introduction

Pesticides, in general, are chemicals used

worldwide in agricultural production to destroy

or control weeds, insects, fungi, and other pests.

Some of those pesticides remain on food as residues. When pesticides are applied improperly,

resulting residues can pose significant health

risks to consumers. To protect U.S. consumer

health, Federal and State programs have been

established to monitor levels of pesticide residues present in domestic and imported food

and fodder and to prevent the marketing of food

containing residues that either exceed specific

levels (known as tolerances) set by the Environmental Protection Agency or for which no

tolerances have been established for that food.

Public concern over pesticide residues in

food has been increasing during the last decade. For example, a recent 1988 national survey by the Food Marketing Institute showed

that approximately 75 percent of consumers are

very concerned about pesticides in their food;

that percentage is higher than that of consumers

worried about cholesterol, fats, salt, additives,

or any other components (2). Contributing to

such concerns have been the discovery of hazardous effects from certain pesticides once

deemed safe, e.g., ethylene dibromide (EDB)

and chlordane, and publicized acute food poisonings from improperly used pesticides, e.g.,

the aldicarb contamination of watermelon in

the Western United States and Canada in 1985.

Finally, the high level of uncertainty concerning the health effects of pesticide residues has

heightened consumer concern.

One factor in this uncertainty is the technical capability of Federal and State programs

to analyze food for all pesticides. These programs are faced with an enormous number of

pesticide/food combinations to test, and the difficulty of the task is compounded by a lack of

information on what pesticides actually have

been used on specific crops (especially for imports). Analyzing for all pesticides on all types

of food products is currently impossible because of limitations in testing methods as well

as time and resource constraints. Although the

number of pesticide/food combinations to address can be narrowed by focusing on the potentiall y moderate to high health hazard combinations, current analytical methods are not

adequate to identify and quantify all residues

of these pesticide/food combinations within

available resources.

Although Federal data show that only a small

percentage of food samples tested violate established tolerances, a gray area exists for those

pesticides and pesticide/food combinations that

are not being analyzed because of the cost or

time of analysis or that cannot be detected by

existing analytical methods. Also included in

this area are a number of pesticides not yet addressed in the monitoring programs, such as

significant pesticide metabolizes, new pesticides, foreign-used pesticides not approved for

use in the United States, and pesticide ingredients categorized as inert. Thus, analytical

methods have become one of the limiting factors in enforcing pesticide tolerances in food.

The uncertainty over this gray area often is interpreted as a lack of proof of the safety of food

and so contributes to public concerns over pesticide residues.

Recent evaluations of Federal pesticide monitoring programs have highlighted the gap between the number of pesticides that could potentially be found in food and the number of

pesticides that can be detected by methods routinely used. Although the size and the public

health significance of this gap are debatable,

a general consensus exists that improved analytical methods could help enhance the effectiveness of monitoring programs.
—

4

Toward this end, increasing interest exists,

including in the U.S. Congress,’ in expanding

the capability of current analytical methods and

developing new methods to detect pesticide

residues in food. Emphasis is being placed on

making methods more practical, e.g., able to

identify increased numbers of pesticides, be less

time-consuming, and use equipment commonly

found in analytical laboratories. Emphasis also

exists on improving methods to address those

pesticide residues with the greatest potential

health hazards. Furthermore, attention is being given to potential applications of new,

emerging analytical technologies, such as new

instrumentation, technologies based on biologically produced reagents, more rapid semiquantitative and qualitative techniques, and technologies that could be easily used outside the

laboratory.

Although increased interest exists in improving analytical methods, no consensus has yet

developed on the importance of doing so. In

contrast to the general public’s uneasiness over

pesticide residues in food, the Federal agencies

responsible for regulating foods do not have

the same level of concern for the situation as

it exists. Based on the low violation rates found

in food under current testing programs, the

Food and Drug Administration (FDA) of the

U.S. Department of Health and Human Services and the Food Safety and Inspection Service (FSIS) of the U.S. Department of Agriculture consider that pesticide residues in foods

is not the most important food safety issue.2

The agencies consider other sources of food

contamination such as microbiological and animal drug residues as having higher priority in

allocation of their resources.

Because of growing congressional interest in

pesticide analytical methods, the Office of Technology Assessment (OTA) was requested by

Congress to examine the state of the art of analytical technologies and methods to detect pesticide residues in food and offer options on how

Federal agencies, especially FDA, could improve their analytical capability through the

adoption or improvement of technologies and

analytical methods. This OTA report provides

a brief assessment of existing, new, and emerging analytical technologies and methods to detect pesticide residues in food. Second, the

report addresses Federal research and programmatic issues relevant to the development and

adoption of technologies and methods.3 Information presented in this report was gathered

from 1) telephone interviews with some 50 experts; 2) visits to 7 pesticide analytical laboratories: 3 FDA, 1 State, 2 private, and 1 foreign;

3) OTA staff research; and 4) a 2%-day OTA

technical workshop. The workshop participants and observers are listed in appendix A

and the 13 peer-reviewed technical papers presented at the workshop are in appendix B.

‘Several bills have been introduced in 1987-1988 of the Iooth

Congress that include sections on the development and implementation of more “practical” analytical methods for the detection of pesticide residues in food.

1.

CHAPTER 1

Food and Drug Administration, Office of the

Associate Commissioner for Legislative Affairs,

official Agency response to OTA June 1988 draft

report, June 29, 1988.

‘FDA believes, and has frequently told the Congress, that the

low incidence of illegal pesticide residues in the American food

supply and the results of FDA’s Total Diet Study indicate that

pesticide residues in food do not pose a health hazard (l).

‘The issue of the actual public health hazard due to pesticide

residues in food was outside the scope of this OTA study.

REFERENCES

2, Food Marketing Institute, Trends: Consumer

Attitudes and the Supermarket (Washington, DC:

Food Marketing Institute, 1988).
Chapter 2

Federal Pesticide Residues in

Food Monitoring Programs
—

Contents

Page

The Legal Basis for Federal Pesticide Monitoring in Foods . . . . . . . . . . . . . . 7

Federal Monitoring Programs . . . . . . . . . . . . . . . . . . . . . . .

Environmental Protection Agency . . . . . . . . . . . . . . . . .

Food arid Drug Administration of the

U.S. Department of Health and Human Services . . .

The Food Safety and Inspection Service of the

U.S. Department of Agriculture . + , . . . . . . . . . . . . . . #

Other Pesticide Residues in Food Monitoring Programs

Chapter 2 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Figures

Figure

2-1. FDA Pesticide in Food Monitoring Program in 1987

2-2 Number of Food Monitoring Samples Analyzed for

pesticide Residues in 10 States in 1987 . . . . . . . . . . .

Tabl e

Table

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2-I. Numbers of Compounds Determined or Identified by

Primary FDA Multiresidue Methods. . . c . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
——

Chapter 2

Federal Pesticide Residues in

Food Monitoring Programs

THE LEGAL BASIS FOR FEDERAL PESTICIDE

MONITORiNG IN FOODS

Comprehensive Federal food laws are a 20thcentury phenomenon, although the States regulated food quality before 1900. The 1906 publication of Upton Sinclair’s novel The Jungle

sparked a consumer reaction against the adulteration and misbranding of food, which resulted

in the passage of the Federal Meat Inspection

Act (FMIA) of 1906 and the Pure Food and

Drugs Act (F&D Act) of 1906 (5). Both statutes

have been significantly amended, although they

retain their original purposes today. The F&D

Act of 1906 was revised into the Federal Food,

Drug, and Cosmetic Act (FFDCA) in 1938. The

origin of Federal regulation of pesticide use can

be traced to this same general period with the

Federal Insecticide Act (FIA) of 1910. The FIA

was replaced by the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) of 1947,

which as amended, remains the basis for regulating the use of pesticides today.

Currently, Federal jurisdiction over pesticide

residues in food is divided among four bodies—

the Environmental Protection Agency (EPA),

the Food and Drug Administration (FDA) of the

U.S. Department of Health and Human Services, and the Food Safety and Inspection Service (FSIS) and Agricultural Marketing Service

(AMS) of the U.S. Department of Agriculture

(USDA). Their authority for this work comes

primarily from five laws: FIFRA, FFDCA, FMIA,

the Poultry Products Inspection Act, and the

Egg Products Inspection Act.

EPA, under FIFRA, must register a pesticide

before the pesticide can be distributed or sold

in the United States (7 U.S.C. Section 136 et

seq., 1982 & Supp. IV 1986), The registration

includes the specific commodities the pesticide

can be used on. In registering a pesticide, EPA

balances the risks and benefits associated with

the use of that pesticide while ensuring that its

use will not cause an unreasonable risk to humans or the environment. ’

If the pesticide is to be used on food or feed

commodities or if its use will lead to residues

on these products, EPA, under FFDCA, establishes the legal maximum level of the pesticide

residue (including residues of significant metabolizes or degradation products) allowed in each

specific food or feed (FFDCA; 21 U.S.C. Sections 346A, 1982 & Supp. IV 1986). These levels

are known as tolerances. A tolerance, or an exemption from a tolerance, must be granted before such a pesticide is registered. Tolerances

cannot be legally exceeded and residues of pesticides are prohibited on foods for which no

tolerance has been established or exempted.

Commodities that violate these prohibitions are

subject to seizure by FDA, USDA, or a State

enforcement agency (33).

FDA, under FFDCA, has responsibility for

enforcing tolerances established by EPA in food

(except meat and poultry) and animal feed moving in interstate commerce and for enforcing

prohibition of a pesticide residue in food or feed

for which no tolerance has been set or exemption given (21 U.S.C. Sections 331-337, 1982 &

Supp. IV 1986).2

‘The registration requirements for pesticides are set forth in

Section 3 of FIFRA and are defined more fully in EPA regulations (40 C.F. R. Sections 158 and 162 1987).

‘In some cases, a specific residue may be present on a commodity for which no tolerance exists because of the environmental persistence of the pesticide rather than its direct application on the commodity. If in this type of case FDA considers

low levels of such a residue to pose little risk to human health,

FDA used to informally set regulatory residue levels called “action levels” at which FDA would take regulatory action and below which the food was not found to be violative (21 CFR Sec-

(continued on next page)

7
—

8

FSIS of USDA is responsible for enforcing

tolerances in meat and poultry under authority of the Federal Meat Inspection Act (21

U.S.C. Sections 601-695, 1982& Supp. IV 1986)

[continued from previous page)

tions 109 and 509, 1987; FDA Compliance Policy Guides, 1986).

The informal process by which these action levels have been

set has been vacated by the Federal Appeals Court in the District of Columbia Consumer Nutrition Institute v. Young, 818

F.2d 943 (D.C. Cir. 1987). EPA and FDA are currently determining how to address these cases.

and the Poultry Products Inspection Act (21

U.S.C. Sections 451-470, 1982& Supp. IV 1986).

AMS of USDA is responsible for pesticide residue monitoring of raw egg products (dried, frozen, or liquid eggs) and tolerance enforcement

at establishments having official USDA egg

products inspection service under authority of

the Egg Products Inspection Act (21 U.S.C.

1031-1056). Under this act, FDA has jurisdiction over these products outside such establishments.

FEDERAL MONITORING PROGRAMS

Environmental Protection Agency

(EPA)

EPA has no direct responsibility for enforcing pesticide tolerances in food; therefore, its

monitoring of pesticide residues in food is minimal. EPA’s primary responsibilities concerning pesticide residues in food, as noted earlier,

are registering the pesticides and establishing

pesticide tolerances for food and feed. EPA’s

pesticide monitoring work is geared primarily

toward regulating pesticide levels in the environment (e. g., water, air, and soil) and ensuring that pesticides are being used in accordance with their registration. However, EPA

conducts some monitoring for pesticide residues in food as part of its monitoring of pesticides in the environment. Agricultural commodities are occasionally analyzed by EPA as

a means of identifying pesticide misuse or pesticide drift from point of application or, if necessary, of obtaining additional residue data for

a pesticide under Special Review to determine

if a pesticide’s registration should be canceled,

denied, or reclassified because of adverse effects (32).

The tolerance-setting process is the basis for

FDA and USDA regulation of pesticide residues

in food. As part of the tolerance-setting process, EPA requires the submission of, among

other things, the following: 1) residue chemistry data, e.g., what residues occur and how

much of each is present; 2) toxicity data; and

3) an analytical method to detect the pesticide

and its toxic metabolizes in the foods for which

a tolerance is to be set. The first two sets of

data are used by EPA to determine the likely

level of dietary exposure to the pesticide, level

of dietary exposure acceptable for human health,

and the tolerance level in each food (33).3 (For

a detailed description of the tolerance-setting

process, see ref. 24.)

Limitations of the tolerance-setting process

may affect the capabilities of FDA and USDA

to monitor for pesticide residues in food. For

example, if incomplete metabolism studies

were used in setting tolerances, then all the possible metabolizes and breakdown products of

the pesticide are not known and methods for

their analysis may not be available or required

(33). A second important limitation (discussed

in chapter 7) is the regulatory usefulness of the

methods submitted to EPA as part of the tolerance setting process.

3The majority of tolerances are established for pesticides on

raw agricultural commodities and set to protect the public health

while considering the benefits of the pesticide use. A small number of tolerances are set for processed foods. Under FFDCA,

if a pesticide concentrates during food processing and therefore occurs in a higher concentration in the processed food than

in the raw agricultural commodity, the decision to establish a

tolerance must be only risk-based, without the consideration of

potential benefits. An additional rule, applying only to processed

foods, is that if a pesticide that concentrates during processing

also causes cancer in humans or animals, then no tolerance can

be granted for the processed food. For pesticides that do not concentrate, the tolerance for the raw agricultural commodity

suffices for processed foods. For further details on this distinction see ref. 25.
9

Food and Drug Administration (FDA)

of the U.S. Department of Health

and Human Services

FDA enforces pesticide residue tolerances

established for a wide variety of raw agricultural food and feed, and for processed products. Commodities sampled do not include meat

and poultry, which are the province of USDA.

To fulfill its regulatory responsibilities, FDA

established a pesticide monitoring program that

is designed to identify and quantify pesticide

residues in food and animal feed. The two main

objectives of this program are: 1) to monitor

domestic and imported food and feed commodities for pesticide residues in support of regulatory actions against illegal residues, and 2) to

gather information on the incidence and levels

of pesticide residues in the food supply (28).

The potential coverage of the FDA’s pesticide

monitoring program includes approximately

316 pesticides for which tolerances have been

established; pesticides whose registrations have

been canceled but persist in the environment;

pesticides previously exempted from the establishment of tolerance levels but for which safety

concerns have subsequently arisen; pesticides

with experimental use permits or pending tolerances; pesticides used only in foreign countries; and metabolizes, other breakdown products, and impurities of pesticide products (28).

Given that the monitoring of all pesticide/

commodity combinations for all of these pesticides would far exceed the resources of the

FDA, a selective monitoring approach has been

adopted (28). The two primary factors used to

determine which pesticide/commodity combinations will be monitored are: 1) analytical

method capabilities, that is, largely the capabilities of multiresidue methods; and 2) priorities of monitoring in terms of the likelihood of

pesticide application to certain commodities

and the potential health risk to the consumer

from consumption of a particular pesticide/

commodity combination (28). The risk assessment is made primarily on the basis of the FDA

Surveillance Index (SI).4 Table 2-1 shows how

much pesticide coverage is provided by the five

major multiresidue methods (i.e., methods that

can detect more than one pesticide during an

analysis of a single sample) routinely used by

FDA to monitor pesticide residues in food.

4At the recommendation of an FDA study group (10), a fivelevel risk classification was developed on the basis of avai]ab]e

toxicological data and potential human dietary exposure. The

categories established are as follows: Class I, pesticides posing

high health hazards; Class II, pesticides posing a possible high

risk; Class III, pesticides posing a moderate hazard; Class IV,

pesticides posing a low hazard; and Class V, pesticides posing

very little potential hazard (29). A complete description of the

process of assigning a pesticide to one of these classes is presented in Reed (29). The Surveillance Index is not yet complete.

Two hundred and five pesticides have been ranked thus far.

Table 2.1.— Numbers of Compounds Determined or Identified by Primary FDA Multiresidue Methodsa

Number of compounds determined or identified

Total Totalb

entered in for all 5 PAM I sec. no.

Type of compounds data base methods 21 1.1/231.1C 212.1/232/.l d 232.3 e 232.4/242/.l f 242.2 g

Pesticides with tolerances . . . . . . . . . . . . . 316 163 68 85 55 140 20

Pesticides with temporary or pending

tolerances . . . . . . . . . . . . . . . . . . . . . . . . . . 74 10 4 3 4 9 4

Pesticides with no EPA tolerance . . . . . . . 56 25 17 21 7 10 0

Metabolizes, impurities, alteration products,

and other pesticide-associated

chemicals . . . . . . . . . . . . . . . . . . . . . . . . . 297 92 20 32 31 61 8

aAs of May 1988.

~his number is not cumulative because several methods may detect the same pesticide.

cGas Chromatographic method for nonpolar (primarily organochlorine and organophosphorus) Pe.stkides in fatty foods,

dGas ~hr~mat~graphic method for nonpolar (p~irna~ilY organochlorine and organophosphorus) pesticides in nonfalty foods.

eGas chromatographic method for organophosphorus pesticides and metabolizes

fGas ~hromatographic method for polar and nonpolar pesticides, USing a variety of Selective detectors.

gLiquidchromatographic method primariiy for N-methyl carbamate pesticides.

honly ce~aln of the chemicals in these four pesticide-related groups necessarily occur as residues or are of toxicolo9icaI concern.

SOURCE D. Reed, P. Lombardo, J Wessel, et al , “The FDA Pesticides Monitoring Program,” Jouma/ of the Associafiorr of Official Arra/ytica/ Chemists 70(3) 593, 1987

The 1987 table was updated for OTA by the Center for Food Safety and Applied Nutrition of the Food and Drug Administration in May 1988,
10

The two major components of the FDA pesticide monitoring program are: 1) general commodity monitoring, and 2) the Total Diet Study.

General Commodity Monitoring

General commodity monitoring is designed

to enable the enforcement of tolerances established by EPA and determine the incidence and

levels of residues in domestic and imported raw

agricultural commodities, processed foods, and

animal feed (28). More specifically, the objectives of this program are to: 1) determine on

a geographical basis pesticide residue levels of

individual food commodities, 2) survey on a

nationwide basis pesticide residue levels of

selected food commodities, 3) monitor imported

food commodities and deny entry to those with

illegal pesticide residues, and 4) identify pesticide residues occurring in excessive levels as

a basis for compliance followup and enforcement action.

Approximately 15,000 commodity samples

were analyzed in 1987 for pesticide residues

by 16 FDA laboratories under the general commodity monitoring program. About 47 percent

of samples were from domestic sources and 53

percent were imported commodities (22; figure 2-1). Emphasis on imported commodities

has increased in the past few years. The majority of samples are collected at random for

monitoring purposes and are known as surveillance samples. The remainder, known as compliance samples, are collected after a violation

has been found or there is evidence of a likely

violation (28). Imports receive more compliance

sampling because less information is available

on foreign growing areas, pesticide use, and

agricultural practices than for domestic commodities (16). FDA’s ability to prevent violative

food from reaching the consumer is constrained

by the amount of time needed for sample transport and analyses. As such, food sometimes is

able to reach the market before results of analyses are available (34). FDA can detain imported

commodities until compliance analyses are

completed but cannot detain domestic commodities (34).

The percentage of samples that violate EPA

tolerances is known as the violation rate. FDA

believes that violation rates cannot be extrapolated to give the correct level of violations in

the general food supply because the biased nature of FDA sampling (both compliance and surveillance sampling) would lead to the calculation of an overly high level of violations (17,

20). First, compliance samples will have a

higher violation rate than the general food supply and the surveillance samples because compliance sampling is done only when a violation

is suspected. Second, surveillance samples are

not conducted in a totally random fashion. Surveillance sampling is biased toward pesticide/

commodity combinations with past residue

problems and also contains a greater percentage of fruits and vegetables than exists in the

general food supply (17).

For all food samples analyzed by FDA in

1987, the violation rate for surveillance samples was 2.5 percent (1.5 percent for domestic

samples and 3.4 percent for imports). The violation rate for compliance samples analyzed

that year was 11.7 percent (12.1 percent for domestic samples and 11.6 percent for imports)

(17). Additional data on violation rates have

been compiled by FDA’s Los Angeles laboratory based on 5 years (1982-1986) of its analysis of almost 20,000 samples (93 percent of

which were surveillance samples and 67 percent were imports). The majority of these samples were fruits and vegetables. The violation

rate for surveillance samples was 2.76 percent

(3 percent for domestic samples and 2,6 percent for imports). The violation rate for compliance samples was 17.8 percent (19.7 percent

for domestic samples and 17.5 percent for imports) (16). Seventy-five percent of the violations

stemmed from pesticide residues on commodities that did not have a tolerance established

for the pesticide (20).

The Center for Food Safety and Applied Nutrition (CFSAN) is responsible for much of the

direction of the FDA monitoring program, primarily through the development of its annual

series of compliance program guidance manuals.
11

Figure 2-1.— FDA Pesticide in Food Monitoring Program in 1987

Domestic import

samples samples

(7,000 annual samples) (8,000 annual samples)

(9096 surveillance; 10% compliance) (80% surveillance; 20% compliance)

Core Regional Selective General Mexican

samples plan survevs* import import

(1,000) (5,000) (1,000) (6,000) (2,000)

A

❑

❑ MRMs for fatty foods

(method 1 and others) Shell eggs, Fruits, High interest Regional selective

milk, cheese, vegetables, etc. chemicals on plan surveys*

local fish, of local selected products (5,000) (1,000)

shellfish importance

❑ ❑ ❑ 00 ❑ mcl ❑ llzz l ❑ ‘::~:;:;~:~~ ~

❑ m

Note that all numbers of samrMes are armroximate Codes for analytical methods refer tO those mOSt often used for analysis of the samr.Nes in anv cateaorv

Abbreviations: MRM = multi risiduemeihod; CPA = chlorophenoxy acid. Definitions’ compliance samPles = samPles collected from shipments for which there was

-e- ,

prior evidence or suspicion of illegal pesticide residues (i.e., subjective samples); su~eillance samPles = samPles collected from shipments for which there was no

prior evidence or suspicion of illegal pesticide residues (i.e., objective samples).

“A combination of special emphasis surveys and headquarters-initiated surveys.

SOURCE B McMahon and J. Burke, “Expanding and Trackincj the Capabilities of pesticide Multi residue Methodology Used in the Food and Drug Administration’s

Pesticide Monitoring Programs, ” Jouma/ of the Association of Official Analytical Chemists 70(6):1073, 1967. The 1987 table Was updated for OTA by the Center

for Food Safety and Applied Nutrition of the Food and Drug Administration in May 1968.

Four types of sampling plans makeup general

commodity monitoring: core samples, special

emphasis surveys, headquarters-initiated surveys, and regional sampling plans (see figure

Z-I, which combines special emphasis surveys

and headquarters-initiated surveys into selective surveys).

Core samples, which must be analyzed by

each district, are identified by CFSAN. Core

samples are of commodities susceptible to environmental contamination and likely to bioaccumulate fat-soluble pesticides (e.g., fish, milk,

dairy products, shell eggs, and feed) (28).

Special emphasis surveys permit each district

to sample two domestic pesticide/commodity

combinations and two imported pesticide/commodity/country-of-origin combinations, CFSAN

develops the list of combinations for selection

by districts, and districts may propose additional combinations subject to CFSAN approval,

These surveys focus on those pesticides neither

adequately measured nor regularly analyzed by

the five multiresidue methods routinely used.

These pesticides may be selected for monitoring because of EPA requests, FDA investigatory reports, a high SI classification for a pesticide, or past violation problems (28).
—

12

Headquarters-initiated assignments (or special surveys) are those in which CFSAN instructs a district to analyze a specific commodity.

Finally, regional sampling plans (for domestic and imported food) allow each region to determine what products it plans to sample based

on its knowledge of local crops, pesticide use,

and coordination with State programs. In 1988,

FDA required each region for the first time to

write up these plans and submit them to headquarters (19).

Ultimately, the number of samples collected

and analyzed for pesticide residues in a district

is determined by the available resources provided by FDA headquarters for pesticide monitoring in that district. FDA laboratories, in

addition to monitoring foods for pesticide residues, also monitor foods for sanitation and

microbiological contamination. Monitoring

nonfood products such as medical devices and

drugs are their responsibility as well. Pesticide

monitoring must compete for resources with

these other significant public health functions,

and sampling plans are sometimes derailed by

emergency situations (e. g., a product tampering incident).

The Total Diet Study

Since the early 1960s, FDA has monitored dietary intake of pesticides in a “market basket”

of selected food items (including meat and poultry) that are purchased at the retail level and

then prepared ready-to-eat prior to analysis.

CFSAN determines the commodities to be sampled, and the analysis is carried out by the FDA

Total Diet laboratory in Kansas City, MO. Two

hundred thirty-four foods selected to represent

the diet of the U.S. population are collected in

retail markets four times annually, once from

each of four designated geographical areas of

the United States (northeast, south, north central, and west) (22, 27, 28). A single collection

consists of identical foods from retail stores in

three cities within each geographical area (27).

Samples are sent to the Total Diet Laboratory,

where the three samples of each food are combined to form a single sample and analyzed

using multiresidue methods (27).

The results of the Total Diet Study (TDS) are

used to estimate dietary intake of selected pesticides by various U.S. age-sex groups (27). The

design of the TDS provides an estimate of public exposure to those pesticide residues detected

by the analytical methods used in the study.

FDA uses data from the TDS to make judgments

about the public health risk presented by pesticide exposure through food (27). In 1987, the

TDS detected 53 pesticide residues out of 253

pesticides detectable by the analytical methods

used. The residues were compared with acceptable daily intakes calculated by the World

Health Organization and none were found to

exceed 1 percent of those acceptable levels (18).

The TDS, however, uses only multiresidue

methods to detect pesticide residues in food.

Therefore, the TDS only provides a partial estimate of total human exposure to pesticide residues in the diet because some pesticides cannot be detected by multiresidue methods.

The Food Safety and Inspection

Service (FSIS) Of the U.S.

Department of Agriculture

The USDA pesticide residue in food monitoring program is part of its National Residue

Program (NRP), which addresses residues of

pesticides, animal drugs, and environmental

contaminants in meat, poultry, and raw egg

products. NRP was initiated by the USDA in

1967 and has grown substantially in terms of

the numbers of samples analyzed. Overall, approximately 50,000 samples are analyzed annually for about 100 compounds (12, 13, 23).

In 1987, the NRP analyzed some 15,260 samples for pesticide residues, and almost 60 percent of these were imported products (15).

The decision of what pesticide to analyze is

based on a ranking of the pesticide (based on

the pesticide’s toxicity and level of human exposure), the capability of testing for the pesticide using a multiresidue method, and past
13

Photo credit: Food and Drug Administration

Samples are collected and shipped to laboratories for

analysis. Above, an FDA inspector samples imported fruit

entering the United States from Mexico at Nogales, AZ.

residue problems with the pesticide (12). Monitoring decisions are based on a list of approximately 227 ranked pesticides and metabolizes

that FSIS considers of potential concern (12).

Similar to the SI developed by FDA, FSIS established the Compound Evaluation System (CES)

in 1985 to improve its ranking of pesticides and

bolster support of its monitoring program and

analytical methods development work. Pesticide residues are assigned a letter rank for their

toxicity (A-D) and a number rank for the degree of human exposure to them (1-4) with A-1

as the highest ranking. Currently, 39 pesticides

have been ranked under the CES (30). An advisory board of scientists from EPA, FDA, and

USDA was also established in 1985 to help keep

pace with new information on compounds of

concern (12).

pesticide residue analysis is accomplished by

using multiresidue methods.5 Normally, a sample is analyzed using one of four multiresidue

methods (for chlorinated hydrocarbons, chlo5FSIS requires that its analytical methods be “practical,”

which it defines as: a] requiring no more than 2 to 4 hours of

analytical time per sample (batches of samples may take longer),

b) requiring no instrumentation not customarily available in a

laboratory devoted to trace drug or environmental analyses, c)

having a minimum proficiency level at or below the established

tolerance, d) having a quality assurance plan, and e) having undergone an interlaboratory validation study (12).

rinated organophosphates, organophosphates,

and carbamates), which together can detect approximately 40 pesticides (1,15). 6 FSIS has

identified 10 highly ranked pesticides it would

like to monitor routinely but cannot using its

multiresidue methods (15). In addition, a number of other highly ranked pesticides exist that

cannot be detected by multiresidue methods but

that FSIS considers less likely to appear in meat

(15). FSIS has three laboratories performing pesticide residue analysis and has contracts with

57 non-Federal laboratories that are accredited

by FSIS to conduct pesticide analyses as well

as analyses of other compounds such as PCBs.

These laboratories are accredited only for the

pesticide analysis of chlorinated hydrocarbons

and they must use an FSIS approved method.

FSIS runs a quality assurance program for these

laboratories using check samples and onsite reviews (9).

The four components of the National Residue Program are monitoring, surveillance, exploratory projects, and prevention, which are

administered by the FSIS (31). Violation rates

for the pesticides analyzed in meat products

are low. In 1986, no violations were found for

the 16 organophosphates tested for in either the

monitoring or surveillance programs. For the

13 chlorinated hydrocarbon pesticides tested

for in 1986, 9 violations out of 3,498 monitoring samples (a 0.26 percent violation rate) and

18 violations out of 1,071 surveillance samples

(a 1.7 percent violation rate) were found (11).

Testing for carbamates began in 1987 and results have not yet been compiled. FSIS believes

that monitoring data can be used to provide a

good indication of violation rates in the general meat supply because monitoring sampling

is random (14). However, monitoring data

would first have to be adjusted for the difference between the number of samples taken

from each animal group and the relative production of each animal to get a proper indication (6). Surveillance data are too biased to be

used the same way (14).

6FSIS also analyzes for eight compounds fed directly to animals as larvicides or to kill insects in animal dung, In 1987, 2,914

samples were analyzed for these products (15),
.

14

The monitoring program involves random

sampling of meat and poultry tissue during routine inspection at slaughter of domestic animals

(FSIS personnel are located at processing plants)

and of imported products at the port of entry.

These samples account for approximately 80

percent (based on 1985 figures) of the total number of samples analyzed (11, 12). The random

sampling scheme used in this program is designed statistically to provide 95 percent assurance of detection over the course of a year with

a violation rate of 1 percent or more in the national population (12). Monitoring samples are

analyzed by the three FSIS laboratories and several of the FSIS accredited laboratories. In most

cases, the FSIS monitoring program does not

prevent violative products from reaching the

consumer because analytical results are not

normally available until after the product has

reached the marketplace and become difficult

to trace (12). Monitoring provides information

on the occurrence of residue violation and helps

to identify those producers who maybe selected

for surveillance sampling because of violations.

The surveillance program focuses on the investigation and control of movement of meat

and poultry products that are suspected of contamination. Unlike the random sampling conducted under the monitoring program, the sampling conducted under the surveillance program

is directed specifically to those meat or poultry carcasses that have been implicated as

sources of residues either by the monitoring

program, by investigation, or by a prior history

of violation by the supplier (31). Carcasses are

held until the analysis is complete. Violative

meat is condemned and the producer is prohibited from marketing animals until further

samples show no illegal residues (26). Analysis of surveillance samples takes precedence

over monitoring samples (12). Analysis may be

done either by the three FSIS laboratories or

else the producer, in order to increase the speed

of analysis, may choose to send a meat sample

to one of the 57 FSIS accredited laboratories

for analysis for chlorinated hydrocarbons. The

producer pays for the analysis, and the results

go first to the FSIS inspector. Unless there has

been a serious contamination event, only a few

hundred surveillance samples are analyzed annually for pesticides by the accredited laboratories (2).

Exploratory projects are surveys used to determine if a pesticide not currently detected

should be included in the monitoring program.

New methods that have not been validated by

FSIS maybe used in these surveys to detect the

pesticides and to evaluate the value of the

method (9).

To complement its regulatory work at the

slaughterhouse, FSIS has a prevention program

based on producer testing and education. Memoranda of Understanding (MOU) are signed

with producers who then pay for testing feed,

feed additives, litter, and some animals for chlorinated hydrocarbons. About 7 FSIS accredited

laboratories perform approximately 2,000 analyses a month and provide FSIS with access to

the results (15). Currently, 11 companies (5 beef

and 6 poultry producers) take part in this program (3). FSIS has also collaborated with the

USDA Cooperative Extension Service to produce educational materials for and provide

counseling to producers on how to avoid chemical contamination of animals.

The AMS carries out a small regulatory program for pesticide residues in raw egg products.

At its laboratory in Gastonia, NC, approximately 400 to 500 samples are analyzed annually from the approximately 90 domestic, eggbreaking and drying factories and imports using

a multiresidue method that can identify 50 pesticides (21). If violations are found in domestic

egg products, AMS may analyze raw eggs to

find which producer is the source of the violative eggs. Both in 1986 and 1987, AMS found

no violations among its monitoring and surveillance samples (21).
15

Although outside the scope of this report,

State and private programs carry out a significant amount of monitoring for pesticide residues in food. Data provided by FDA to the General Accounting Office (GAO) showed that 38

States had such monitoring programs (34). State

programs vary widely in the number of samples processed and in the program purpose. ’

For example, Montana’s and Florida’s programs

focus on the most likely cases of overtolerance,

e.g., if there has been a major pest outbreak that

could lead to overuse of a pesticide; Massachusetts has directed its program to dietary risks

(4), Figure 2-2 provides a survey of 10 State pro7For more information about State programs, see Cusick and

Wells, 1988 in appendix B.

grams and the number of monitoring samples

analyzed for pesticide residues. State programs

rely primarily on multiresidue methods,

The extent of private sector testing is more

difficult to determine. A considerable amount

of monitoring by food processors is taking place

but remains proprietary information, in part

because of fears of the possible negative connotations associated with such testing (7). An

example of this work is the National Food Processors Association, which estimates it analyzes

approximately 3,000 food samples a year for

pesticide residues for its members (8). Federal

agencies are interested in using private monitoring data, partly to help set their own monitoring priorities, and EPA has an ongoing project to collect the results of private monitoring,

Figure 2-2.— Number of Food Monitoring Samples

Analyzed for Pesticide Residues in 10 States in 1987

. . ---

14,W U

13,000 -

12,000 “

11,000 -

10,OOO -

9,000 -

8,000 –

7,000 –

6,000 –

5,000 -

4,000 –

3,000 –

2,000 –

1,000 -

0 —

Photo credit: California Department of Food and Agriculture

When a widespread pesticide/commodity problem occurs,

the California Department of Food and Agriculture

may send one of its three mobile laboratories to

assist with monitoring.

CA FL TX NY OR WA MA AZ HI MT

SOURCE” California Department of Food and Agriculture, Pesticide Enforcement

Branch. Survey done for OTA, 1988.
—.

16

1. Ashworth, R., Chemistry Division, Food Safety

and Inspection Service, Washington, DC, personal communication, May 13, 1988,

Z. Barth, J., Chemistry Division, Food Safety and

Inspection Service, Washington, DC, personal

communication, June 29, 1988.

3. Cielo, A., Residue Evaluation and Planning Division, Food Safety and Inspection Service,

Washington, DC, personal communication,

July 7, 1988.

*4. Cusick, W., and Wells, J., “Pesticide Analytical Methods Development at the State Level, ”

OTA commissioned paper, Spring 1988.

5. Dingel, J. D., “One Goal, Many Agencies: An

Assessment of Pesticide Residue Regulation in

Animal Feeds and Animal Meat Products,”

Food, Drug, Cosmetic Law J. 41:467-511, 1986.

6. Elder, R., Mathematics and Statistics Division,

Food Safety and Inspection Service, Washington, DC, personal communication, July 28,

1988.

7. Elkins, E., National Food Processors Association, Washington, DC, personal communication, Apr. 20, 1988.

8. Elkins, E., National Food Processors Association, Washington, DC, personal communication, Apr. 6, 1988.

9. Ellis, R., Chemistry Division, Food Safety and

Inspection Service, Washington, DC, personal

communication, July 7, 1988.

10. Food and Drug Administration, Study Group

on FDA Residue Programs, “FDA Monitoring

Programs for Pesticide and Industrial Chemical Residues in Food” (Rockville, MD: Food

and Drug Administration, June 1979).

11. Food Safety and Inspection Service, U.S. Department of Agriculture, Domestic Residue

Data Book, National Residue Program 1986

(Washington, DC: Food Safety and Inspection

Service, October 1987).

12. Food Safety and Inspection Service, U.S. Department of Agriculture, Compound Evacuation and Analytical Capability National Residue Plan, 1987 (Washington, DC: Food Safety

and Inspection Service, January 1987).

13. Food Safety and Inspection Service, U.S. Department of Agriculture, “FSIS-18, Facts: The

National Residue Program I“ (Washington,

DC: Food Safety and Inspection Service, revised January 1984),

*These references are contained in Appendix B.

14. Harr, R., Residue Planning and Evaluation Division, Food Safety and Inspection Division,

Washington, DC, personal communication,

July 27, 1988.

15. Harr, R., Residue Planning and Evaluation Division, Food Safety and Inspection Service,

Washington, DC, personal communication,

June 27, 1988.

16, Hundley, H., Luke, M., Cairns, T., et al., “Pesticide Residue Findings by the Luke Method

in Domestic and Imported Food and Animal

Feeds for Fiscal Years 1982 -1986,” J. Assoc.

Off. Anal.. Chem., forthcoming, 1988.

17. Lombardo, P., Pesticide and Industrial Chemicals Branch, Center for Food Safety and Applied

Nutrition, Food and Drug Administration, Washington, DC, personal communication, July 27,

1988.

18. Lombardo, P., Pesticide and Industrial Chemicals Branch, Center for Food Safety and Applied

Nutrition, Food and Drug Administration, Washington, DC, personal communication, July 13,

1988.

19. Lombardo, P., Pesticide and Industrial Chemicals Branch, Center for Food Safety and Applied

Nutrition, Food and Drug Administration, Washington, DC, personal communication, July 8,

1988.

20. Luke, M., Masumoto, H., Cairns, T., et al.,

“Levels and Incidence of Pesticide Residues

in Various Food and Animal Feeds Analyzed

by the Luke Multiresidue Methodology for Fiscal Years 1982 -1986,” J. Assoc. Off. Anal. Chem.

71(2):415-433, 1988,

21. Magwire, H., Agricultural Marketing Service,

U.S. Department of Agriculture, Washington,

DC, personal communication, May 1988.

22. McMahon, B. and Burke, J., “Expanding and

Tracking the Capabilities of Pesticide Multiresidue Methodology Used in the Food and

Drug Administration’s Pesticide Monitoring

Programs,” J. Assoc. Off. Anal. Chem. 70(6):

1072-1081, 1987.

23, Middlekauff, R. D., “Pesticide Residues in Food:

Legal and Scientific Issues,” Food Drug and

Cosmetic Law J. 42:251-264, 1987.

24. Moore, J., “Statement Before the Committee

on Agriculture, Nutrition, and Forestry, U.S.

Senate,” Federal Insecticide, Fungicide, and

Rodenticide Act, hearings before the Committee on Agriculture, Nutrition, and Forestry,

Senate, US. Congress, May 20, 1987, serial No,

S. Hrg. 100-394, Part I (Washington, DC, 1988).
17

25. National Research Council, Board on Agricul- 304

ture, Committee on Scientific and Regulatory

Issues Underlying Pesticide Use Patterns and

Agricultural Innovation, Regulating Pesticides

in Food: The Delaney Paradox (Washington, *31,

DC: National Academy Press, 1987).

26. National Research Council, Food and Nutrition Board, Committee on the Scientific Basis

of the Nation’s Meat and Poultry Inspection 32.

Program, Meat and Poultry Inspection: The

Scientific Basis of the Nation Program (Washington, DC: National Academy Press, 1985).

27, Pennington, J. and Gunderson, E., “History of *33.

the Food and Drug Administration’s Total Diet

Study,” J. Assoc. Off. Anal. Chem. 70(5):772-

782, 1987.

28. Reed, D., Wessel, J., Burke, J., et al., “The FDA 34.

Pesticides Monitoring Program,” J. Assoc. Off.

Anal. Chem. 70(3):591-595, 1987.

29. Reed, D., “The FDA Surveillance Index for Pesticides: Establishing Food Monitoring PrioriRubin, L., Residue Evaluation and Planning Division, Food Safety and Inspection Service,

Washington, DC, personal communication,

May 16, 1988.

Taylor, S., “Developing Pesticide Analytical

Methods for Food: Considerations for Federal

Policy Formulation, ” OTA commissioned paper, Spring 1988.

Trichilo, C., Office of Pesticide Programs, U.S.

Environmental Protection Agency, Washington, DC, personal communication, June 27,

1987.

Trichilo, C. and Schmitt, R., “Federal Pesticide

Monitoring Programs: Analytical Methods Development,” OTA commissioned paper, Spring

1988.

U.S. Congress, General Accounting Office, “Pesticides: Need to Enhance FDA’s Ability to Protect the Public From Illegal Pesticides, ” GAO/

RCED-87-7 (Washington, DC: U.S. Government

Printing Office, October 1986).

ties Based on Potential Health Risk, ” J. Assoc.

Off. Anal. Chem. 68(1):122-124, 1985. *These references are contained in Appendix B,
Chapter 3

Contemporary Analytical Techniques

for pesticide Residues in Food
Page

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24

27

29

● ☛☛☛✎ ☛✎☛☛ ☛☛☛☛ **o* *o. *O. * 31
Chapter 3

Contemporary Analytical Techniques for

pesticide Residues in Food

Pesticides may occur in foods in concentrations called trace levels. Trace levels are generally at concentrations of parts per million,

that is, one microgram of pesticide per gram

of food or less. Measuring such small amounts

of pesticides in the presence of enormous

amounts of other chemicals that occur naturally

in food is a challenge because those chemicals

may interfere with measurement, A variety of

analytical methods (see ch. 6) are currently used

to detect pesticide residues, and all contain certain basic steps in application. The basic steps

of an analytical method include the following:

sample preparation: preparation of the

sample to be analyzed by chopping, grinding, or separating plant parts;

extraction: removal of a pesticide residue

from the sample’s other components;

cleanup (isolation): removal of constituents

that interfere with the analysis of the pesticide residue of interest, this step includes

partitioning and purification;

determination–separation: separation of

●

components, individual pesticides, and

sample coextractives according to differential partitioning between a solid or nonvolatile solvent and a liquid or gas carrier that

moves through a column (liquid and gas

chromatography) or along a coated plate

(thin layer chromatography); and

determination—detection: production of a

response that measures the amount of the

components moving through the column,

allowing detection and quantification of

each pesticide.

How these steps interact within any particular method is shown in figure 3-I (l). The cleanup step in figure 3-1 has two parts, partitioning and purification, and the extracting solvent

is either acetone or acetonitrile.

This chapter describes existing and new technologies currently used to analyze pesticide

residues in food and notes how these technologies can improve the analytical steps described

above.

The first step to analyzing a food sample is ite sample from which one or more subsamples

to chop, grind, or otherwise separate plant or

animal parts. The samples must be handled in

such a way as to avoid the loss of volatile pesticide residues and to prevent contamination of

the sample with other pesticides or interfering

chemicals. If only the edible portion of the sample is to be analyzed, it must be removed from

non-edible portions. If several different edible

portions of a food are analyzed separately, the

portions must be separated from each other in

each sample. If, however, several samples are

combined to provide a representative composare to be taken for analysis, all samples must

be handled in an identical manner to avoid inaccurate results in analyzing the subsamples.

Chopping or grinding followed by blending and

mixing are manipulations designed to produce

a homogeneous composite sample from which

subsamples can be taken and to disrupt the

gross structural components of the food to facilitate extracting pesticides from the sample.

Performing this step can be time-consuming

and labor intensive.

21
22

Figure 3-1. -Slmpllfled Scheme of the Steps in the Analysis of Plant Material for

Sample preparation

Extraction

Clean-up

Partitioning

Physical separation of food parts

Combination of samples

Chopping/blending

I 1

I

[ 1

Acetone Acetonitrile

Water

Organic phase

I

[ I ■ 1

Adsorption Gel

Purification column Other No permeation

chromatography chromatography procedures clean-up

I I I

I I 1’

Determination

Separation GLC TLC HPLC Other

Detection

procedures

SOURCE: Modified from A Ambrus and H P Thler, “Allocation of Multiresidue Procedures m Pesticide Residue Analysis,” Pure and

A#Med Chemstr)r 56(7): 1035-1062, July 1986

EXTRACTION

Extraction is performed with a solvent to remove the pesticide residue of interest from

other components of the sample. In most analytical laboratories, a solvent such as acetone

or acetonitrile is used to extract pesticides from

250 grams or less of the food to be analyzed.

The solvent is blended with the food, and smaller

amounts can be further homogenized using an

ultrasound generator. Salts, such as sodium

chloride or sodium sulfate, can be added to absorb water. Or additional water can be added,

if desired, so that the resulting aqueous solution can be partitioned with a water-immiscible

solvent in a subsequent cleanup step.

Extraction times vary from a few minutes to

several hours, depending on the pesticide to

be analyzed and the sample type. Problems that

occur during the extraction process include incomplete recovery and emulsion formation. Incomplete recovery generally can be remedied

by selecting a more efficient solvent. Emulsions,

the production of a third phase or solvent layer

that confuses the partitioning process, can usually be broken down by adding salt to the sample/solvent combination. Residual amounts of

the extracting solvent or partitioning solvent

should not be allowed to reach the detector if

it is an element-specific detector and the sol-
23

vent contains that specific element. These problems can be solved by proper solvent selection

or by removal of the interfering solvent during

the cleanup process.

Supercritical fluids (SFs) may provide a new

technique for extracting pesticides. SFs are

fluids that are more dense than gases but less

dense than liquids. SFs are not yet used in regulatory methods to analyze pesticide residues

in food but are gaining favor among analytical

chemists and food engineers for the ability to

extract a wide variety of chemicals from many

sample types. SFs have many advantages over

conventional solvents. They yield high recoveries of the extracted chemical in a short time,

sometimes as quickly as 10 to 30 minutes at temperatures only slightly above ambient (40 to

500 C). Such temperatures prevent thermal

decomposition of the extracted chemical. Since

some degree of extraction selectivity can be created by choosing an appropriate pressure, this

feature may allow the chemist to separate compounds that may interfere during extraction.

Removal of the SF from the dissolved chemical in the gas form is easily accomplished (19).

The residual chemical of interest can then be

dissolved in a conventional solvent and carried

through one of the conventional chromatographic

analyses (discussed later). Much remains to be

done to explore the usefulness of supercritical

fluid extraction (SFE) for rapidly and efficiently

extracting pesticides from foods.

CLEANUP

Cleanup or isolation removes the constituents

that interfere with the analysis of the pesticide

residue of interest. Cleanup is usually achieved

by a combination of partitioning’ and purification, and the latter is usually accomplished

by preparative chromatography. The degree of

cleanup required is determined by the efficiency with which the partitioning solvent can

remove pesticides from the sample extract

while leaving behind mutually occurring interferences, Special modification techniques may

improve the efficiency of cleanup as well as

the efficiency of detection (16).

The preparative chromatography typically

used for purification is of the: 1) adsorptive, or

2) gel permeation (or size exclusion) type. Adsorption chromatography is based on the interaction between a chemical dissolved in a solvent and an adsorptive surface. Particles of the

chromatographic material are placed in large

glass columns (30 cm x 2 cm), the sample is deposited in a solvent on the top of the column

and eluted with various types of organic sol-

‘Partitioning is the process of distributing the pesticide between two immiscible solvents so that the pesticide will appear

in one phase and potential interferences in another, which then

can be discarded.

vents.

elutes

Separation occurs when the pesticide

in fractions different than the sample

coextractives. Table 3-1 summarizes the materials that have been used with these two types

of preparative chromatographic modes, giving

some of their distinguishing features.

Gel permeation (or size exclusion) chromatography is a technique that separates compounds

from one another on the basis of differences

in molecular size. Preparative-sized columns

similar to those used in adsorption chromatography are used, and samples are placed at the

top of the column and then eluted with a solvent; larger molecules elute before smaller ones

in an ordered fashion. The ordering by size in

gel permeation is a result of small holes designed into the particles placed in the column

that retard the movement of smaller molecules

through the column; such sizing cannot occur

on adsorption columns.

The advantages of gel permeation over adsorption chromatography are that no loss of

pesticide occurs on the column, either by irreversible adsorption or by chemical reactions.

A disadvantage is that a medium-pressure pistontype pump is required to deliver solvent to the

column, making a sample injection valve nec-
24

Table 3-1.—Materials Used for the Preparative

Chromatography of Pesticide Residues in Food

Florisil

1.

2,

3.

4.

5.

6.

A diatomaceous earth adsorbent; retains some lipids preferentially; particularly suited for cleanup of fatty foods.

Good for cleanup of nonpolar pesticides, such as the chlorinated hydrocarbons; produces very clean eluants, removes most interferences when eluted with nonpolar

solvents.

Difficult to use for fruits and vegetables when moderately

polar to polar pesticides are present.

Prone to vary from batch to batch.

Sometimes oxidizes organophosphates with thio-ether linkages; adsorbs some oxons irreversibly.

Most widely used material in the United States.

Alumina

1. Basic alumina can be substituted for Florisil for the cleanup of fatty foods.

2. Does not vary from batch to batch as much as Florisil,

3. Will decompose some organophosphates.

4. Does not effectively separate some plant materials from

the pesticide.

Silica gel

1. Particularly useful for isolation of certain polar pesticides

without losses.

2. Will not adequately separate some plant coextractives from

some pesticides.

3. Will separate some organochlorine pesticides from animal

fat well enough to permit thin layer chromatography.

Carbon

1. Unlike other absorbents, carbon has different elution characteristics due to its lipophilic nature; absorbs preferentially nonpolar and high molecular weight pesticides.

2. Removes chlorophyll well from vegetables but not waxes.

3. Strongly affected by pretreatment; results in literature often

not comparable.

4. Difficult to maintain flow rates in columns.

SOURCE: Office of Technology Assessment, 1988.

essary. The required equipment is more expensive than that used in adsorption chromatography, although such equipment is available in

an automated package.

The cleanup step is often a limitation in pesticide residue methods because it generally consumes a large amount of the total analysis time

and restricts the number of pesticides that are

recovered in some cases, as a result of losses

in chromatography, partitioning, and other

cleanup steps. New technologies such as solid

phase extraction (SPE) (also known as accumulator or concentrator columns) can speed up

cleanup as well as extraction. The SPE packing materials or cartridges retain the pesticide

when the extract is passed through without retaining potential interferences extracted from

Photo credit: Food Safety and Inspection Service Laboratory, Athens, GA

After putting the sample through an alumina packed

column, solvent is added to elute the pesticides off

of the packing in the column.

the food. The SPE cartridge is a small plastic,

open-ended container filled with adsorptive

particles of various types and adsorption characteristics. The pesticide is then eluted and carried forward to an appropriate determinative

step. Conversely, SPE may be used to cleanup

the extract by retaining coextractives and allowing the pesticide to pass through.

SPE technology is particularly attractive for

use in pesticide residue analytical methods,

since it often eliminates the need for expensive

and environmentally sensitive solvents. These

cartridges also have the following advantages:

batch sample processing capabilities, small size,

adaptability to robotic technology, low cost, and

ready availability from many sources. SPEs

have the disadvantages of being unproven for

many pesticides, unable to handle large sample sizes, and generally are ineffective for extracting water-soluble pesticides and metabolites. As more types of absorbents become

available, however, the last disadvantage may

be remedied. Using this type of cleanup sometimes results in losses of pesticides that cannot then be determined chromatographically,

but under such circumstances, cleanup steps

should be minimized or eliminated. SPE is be-
25

ing used by industry and private laboratories

but is not yet routinely used by regulatory agencies to a significant extent, SPE cartridges are

being used by several FDA laboratories to cleanup extracts before the detection step to protect

the column used in high performance liquid chromatography (HPLC).

DETERMINATION-CHROMATOGRAPMK SEPARATION

After a pesticide has been extracted and isolated from the sample, it is further separated

from other coextractives, usually by either gas

chromatography or liquid chromatography or,

less frequently, by thin layer chromatography.

Gas Chromatography (GC)

Separations

Historically, gas chromatography has been

the dominant technique of separation, with at

least 40 years of development and refinement.

Most multiresidue methods (MRMs) used by

the FDA and USDA and most single residue

methods (SRMs) are based on GC.

Separations of pesticides and sample coextractives occur in analytical columns within a

gas chromatography; the columns are usually

made of glass and are either the type that is

packed or the wall-coated, open tubular type

known as capillary.

A column filled with particles is called a

packed column and has an internal diameter

of about 2 millimeters; a column with a thin

film on the wall and an internal diameter of

about 0.1 millimeter is called a capillary column. Packed columns are typically 2 meters

or less in length whereas capillary columns are

typically 10 meters and longer, sometimes

reaching 50 meters.

A sample of food extract, about 10 microliters

or less and either cleaned up or not, is placed

at the beginning of the column where the solvent is flash evaporated along with the pesticide. A gas, called the carrier gas, is continually flowing through the column, moving the

pesticide along, which partitions between the

particles packing the column or the thin film

of involatile liquid on the wall of the column

if it is a capillary column. The relative affinity

of the pesticide for the particles or the thin film

determines when it elutes from the column, at

which time it goes through the detector where

a response is generated and printed out on a

recorder. The continuous trace of such responses is called the chromatogram (figure 3-

2), Chromatographic peaks appear on the chromatogram; their position on the chromatogram

is called the retention time. Quantifications are

performed by measuring the area under the

peak and comparing its area to that of varying

amounts of analytical standards (figure 3-3).

Historically, the packed column has been used

by most pesticide residue analytical chemists.

As a result, a vast amount of retention data’ exists for pesticides on packed columns. One way

of expressing retention data is the use of “relative retention time” (rrt) for a particular pesticide/column combination. The rrt values are

then used to identify an unknown by comparing the rrt to that of a standard. Chlorpyrifos

is typically used as the standard for chlorinated

hydrocarbon and organophosphate pesticides.

The lack of rrt data for capillary columns is a

constraint to their use.

Until the mid-1970s, capillary chromatography was used only when packed columns could

not fully resolve the many components in the

sample undergoing analysis, Today, the availability of a varied and growing selection of

capillary columns has increased their popularity, A conventional capillary chromatogram has

been more time-consuming to develop (requiring as much as 30 to 45 minutes) than packed

column chromatograms (requiring less than 30

minutes) (3). However, the availability of the

~Retention data are retention time (time required to elute a

compound from a chromatographic column) and retention volume (volume of carrier gas required to elute a compound from

a GC column].

87-8.27 0 - 88 - ~
26

Figure 3-2.-Schematic Diagram of a Gas Chromatographic System

Thermostats

1 - - 1 )

I I j

1 I

Recorder Chromatogram

Flow

controller Column

Carrier

gas

bottle

Enlarged cross section

SOURCE: H M McNair and EJ Bonelli, 8asic Gas Chrmra~g~ (Berkeley, CA: Ccmsolidated Printers, 1969)

wide bore capillary column has reduced time.

Table 3-2 summarizes retention data for seven

pesticides on a packed column and on a wide

bore capillary column.

A new generation of hardware gives flexibility in the use of “guard columns, ” pieces of

deactivated but uncoated fused silica tubing

used to guard the analytical column from contamination by deposition of involatile food

coextractives. Such guard columns could enable capillary column chromatography of relatively unclean food extracts that heretofore

could only have been chromatographed on

packed columns (2).

High Performance Liquid

Chromatography (HPLC) Separations

HPLC for the analysis of pesticide residues

is a fairly recent occurrence, but it is becoming the second most frequently used technique

after GC. GC depends upon the volatilization

of the pesticide, whereas HPLC is dependent

on the ability of the chemical to be dissolved

in a suitable solvent.

Separations occur on the analytical column

packed with uniformly sized and shaped particles with a liquid film of varying polarities

or adsorptive sites. A small volume of sample

is deposited on the top of the column, and solvent is pumped through at high pressure. As

the solvent moves through the column, the pesticide distributes itself between the particles

(stationary phase) and the solvent (mobile phase);

the pesticides that have a higher affinity for the

stationary phase exit the column last (figure 3-4).

Stationary phases are commercially available

that can selectively retain any molecular structure—polar, nonpolar, ionic, or neutral; separations can even be made to occur as a function

of molecular size (gel permeation). Chemical

derivatizations, the synthesis of a chemical

derivative of the pesticide, therefore are not required for separations by HPLC. They are used

to label molecules that do not respond to con-
27

Figure 3-3. -Chromatogram of a Gas Chromatography

5 10 15 20

Minutes

During sample analysis, the results of GC and HPLC chromatographic separation and detection steps appear on the chromatogram as peaks. The time it takes a peak to appear is used to

identifv the pesticide. The quantity of the pesticide can be determined-by measuring the area under the peak.

SOURCE: Alltach Associates, Inc, Applied Science LeJM, State College, PA, 1988

ventional analytical detectors. Such labeling

usually enhances the detectability of the molecule. Sometimes labeling is done “post column, ”

i.e., after elution from the chromatographic

column, as for the N-methylcarbamates and carbamoyl oximes (13). Such labeling permits

measurements of these classes of pesticides in

the presence of other potential interferences

as a result of the specificity of the reaction.

HPLC is not as efficient as capillary gas chromatography for separator purposes because

the chromatographic peaks are broader. However, HPLC columns are more efficient than

packed GC columns when columns of equal

Table 3-2.—Comparison of Retention Data for Seven

Chlorinated Hydrocarbon Pesticides on a Packed

Column and on a Wide Bore Capillary Column

Retention times (minutes)b

Pesticidea

Packed columnc Capillary columnd

Lindane . . . . . . . . . . . . 2.5 0.7

Heptachlor . . . . . . . . . 3.9 1.1

Aldrin. . . . . . . . . . . . . . 4.8 1.5

Heptachlor epoxide. . 6.7 1.9

Dieldrin . . . . . . . . . . . . 9.9 2.8

Endrin . . . . . . . . . . . . . 12.0 3.2

P,P’ DDT . . . . . . . . . . . 14.1 4.5

Total analysis

time e 

. . . . . . . . . . . . 15.0 4.7

aBoth columns exfllbit comparable resolution between peaks, R =s > 1.0.

bRetentlon time (time required to elute a compound frOm a chromatographic

column) and retention volume (vOIIJme of Carrier gas required to elute a compound from a GC column).

CGla~g column, 1,8 M x 0.4 cm; PT 40/. SE-30 + 870 OV-21O On Gas Chrom Q,

80/100; 2000C; electron capture detector; nitrogen carrier, flow 90 ml/min; 2 x

10-9 grams for each pesticide.

dopen tubular column, 10 M x 0.053 cm; RSL/200, 1.2 microns thick; 200 CC; electron capture detector; nitrogen carrier, flow 5 mllmin, 15 mllmin makeup;

unknown amounts of pesticide. eRepreSerltS time at Whlcfl all pesticides have pSSS~ through the Column.

GLOSSARY: Gas Chrom Q—a white diatomaceous earth that has been screened,

acid and base washed, neutralized, and silanized (support for liquid phase);

OV-210—50°/0 trifluoropropyl, methyl SiliCOne (liquid phase of gas chromatographic column packing material); PT—pretested; R=s l.O—Resolution (the true

separation of two consecutive peaks) of greater than 1 second; RSL/2W-poIy -

diphenyldimethylsiloxane (liquid phase); SE.20-metIM silicone gum (liquid

phase of gas chromatographic column packing material).

SOURCE: Alltech Associates, Inc. “Cata109 4150,” Avondale, PA, 1988,

Figure 34.-Schematic Diagram of a High

Performance Liquid Chromatographic System

Filter

Drain

valve

solvent - Pump ‘

Pulse

reservoir dampener

Fitter Sample

valve

t

Waste

recycle I I

SOURCE: J M Miller, An/nfroducdonfo LiquidChromefography forthe Gas Chmnmfographw (Bridgewater, NJ: GOW-MAC Instrument Company)
28

length are considered. HPLC columns usually

last longer because they are not subjected to

the extremely high temperatures that GC columns are.

Thin Layer Chromatography (TLC)

reparations

This technique is based upon partitioning a

pesticide between a solvent and a thin layer of

adsorbent, which is usually silica or alumina

oxide that has been physically bonded to a glass

or plastic plate. Samples are applied in a solvent as spots or bands at one edge of the plate

and the plate is then placed in a tank containing a solvent. The solvent migrates up the plate

by capillary action, taking the pesticide with

it and depositing it at a given distance up the

plate. The time required for TLC plate development may range from a few minutes to several hours depending on the pesticide, the solvent, and the adsorbent. Following complete

development, the plate is then removed from

the tank and the spots or bands left by the migration of the solvent are detected using one of

several techniques.

As a separator technique, TLC is much less

efficient than either GC or HPLC because the

resolution separated by TLC is approximately

less than one-tenth of that found using a packed

GC column to produce the same separation.

Consequently, TLC as a separator technique

has largely been replaced by GC and HPLC, On

the other hand, interest exists in using TLCS

to develop rapid, semiquantitative methods (see

ch. 6).

Supercritical Fluid Chromatography

(SFC) Separations

SFS may provide a new technique for chromatographic separation in the regulatory analysis of pesticide residues in food. with SFs as

the solvent phase, SFC can chromatography

chemicals that cannot be handled by gas chromatography because of their involatility or thermal instability. Because the chemical undergoing chromatography diffuses more readily

in the SF than in the liquid used for HPLC, the

solvent can be pumped at a higher velocity, resulting in shorter analysis times. A fringe benefit is that many detectors designed for GC can

also be used in SFC. Detectors that have been

shown to be effective are the flame ionization,

the nitrogen-phosphorus, and the atomic emission spectrometric as well as the UV absorbance detector.

Extraction and chromatographic separation

using SFS was recently demonstrated for the

analysis of sulfonylurea herbicides (8). This

technique, called SFE/SFC, was capable of producing chromatographic responses from extracts

of sand, soil, wheat kernels, whole wheat flour,

wheat straw, and from a cell culture medium.

No recoveries or concentration levels were

given.

Such a coupled extraction and analysis using

supercritical fluids warrant further examination as a rapid means of analyzing pesticide

residues in foods, if automation in general and

robotics in particular can be used for sample

insertion into the instrumentation.

DETERMINATION-DETECTION

This final step of analysis produces a re- Gas Chromatography Detectors

sponse that can be used to measure the amount

of pesticides moving in the column. There are Concurrent with improvements in gas chronumerous types of detectors. These detectors matographic column technology have been maoperate under various principles and have the jor improvements in detectors. These improvability, in some cases, to detect only certain ments resulted in a growing number of detector

classes of chemicals. types becoming available, increases in detec-
29

tor sensitivity due to improved design and enhanced electronic stability, and a trend toward

detector miniaturization, which makes them more

amenable for use in capillary chromatography.

Historically, only five detectors have been

used, They are the electron capture detector

(EC D), Hall microelectrolytic conductivity detector (HECD), the thermionic detectors (NPD

and AFID), and the flame photometric detector. Table 3-3 summarizes their characteristics.

Of these detectors, the ECD was the first to

be used for pesticide residue analysis. ECD

measures the loss of detector electrical current

produced by a sample component containing

electron-absorbing molecule(s). Being very sensitive for measuring halogenated pesticides, its

value lies in the analysis of chlorinated hydrocarbon pesticides such as aldrin, dieldrin, and

DDT. Its sensitivity to such compounds has

made ECD attractive for the analysis of polychlorinated biphenyls (PCBs) as well. ECD also

responds to portions of organic molecules,

other than halogens, which have a large electron affinity, and for that reason the detector

sometimes has difficulties analyzing some unclean crop extracts. Recent improvements in

related electronics and the incorporation of a

high-temperature radioactive source have made

the technology less susceptible to fouling from

crop coextractives.

The Hall detector can be set to measure chlorine (and other halogens), nitrogen, or sulfur.

When set for chlorine, the detector is especially

useful for simplifying the detection of halogenated pesticides because nonhalogens are not

detected, thus producing a simpler chromatogram to interpret. Similarly, when the detector is set for one mode, it will not detect pesticides that require one of the other settings. This

detector is more selective than the ECD, but

the ECD is more sensitive. In addition, the Hall

detector does not need as clean an extract as

the ECD, and therefore its use can lead to faster

methods by allowing reductions in cleanup. A

drawback is that the Hall detector requires

more maintenance than the ECD.

Somewhat less sensitive than the ECD, but

essentially nonresponsive to nonhalogenated

compounds, the Hall electrolytic conductivity

detector also has improved over the last few

years. In fact, it has replaced the ECD in some

laboratories where extreme sensitivity is not

required. The Hall detector can also be set up

for nitrogen and sulfur containing compounds.

Both the NPD and AFID measure the presence of nitrogen and phosphorus atoms in the

pesticide, with little response resulting from

other types of atoms in the molecules. At this

time, the NPD has all but replaced the AFID

in most residue laboratories due to its more simTable 3-3.—Gas Chromatography Detectors Used for Pesticide Residue Analysis

Approximate limit Sample Examples of

Type Selectivity of detection destruction Reliability pesticides detected

Alkali Flame (AFID) Organic P, N 10-12 g P Yes Fair Triazine herbicides

1 0

-10 g N (atrazine)

Electron Capture Detector (ECD) Electronegative 1O-l3 g Cl/see as Iindane Yes Fair Organochlorines

Containing Groups (methoxychlor)

Flame Photometric Organic P, S 10--12 g P/see Yes Excellent Organophosphate

2X10-12 g S/see (malathion)

Hall Electrolytic Conductivity Detector Organic Cl, S, N 1 -2x10-13 g CL/see Yes Fair Organochlorlnes

5-10x10-13 g S/see (aldrin)

1-2x10-12 g N/see

Nitrogen-phosphorus Detector (NPD) Organic P, N <0.2x10 -12 g P/see Yes Good Organophosphates

<0.4X10-12 g N/see (parathion)

Mass Selective Detector (MSD); Ion Trap

Detector (ITD) everything except carrier 10-11 g; MID Yes Good All pesticides

SOURCE Office of Technology Assessment, 1988
30

ple operation as well as more reproducible responses from individual detectors.

The flame photometric detector measures sulfur or phosphorus. It is a rugged detector, highly

stable, and very selective since it does not detect compounds other than organophosphates

and those containing sulfur. The flame photometric detector is less sensitive for phosphorus than the NPD and less sensitive for sulfur

than the Hall detector. However, it is useful for

the analysis of unclean food extracts.

Conventional mass spectrometers (MS) have

been used by some pesticide residue laboratories as gas chromatography detectors and to

a lesser extent as high performance liquid chromatography detectors. Their cost ($150,000 and

higher) has limited their use. MS is normally

used when special techniques are necessary to

confirm the identity of a particular pesticide,

when conventional detectors cannot detect the

pesticide, or for unidentified analytical responses (discussed in ch. 6). Usually an MS is

set to the multiple ion detection mode in order

to gain sensitivity; the alternative would be to

obtain full spectral scans on each chromatographic peak, which is always less sensitive.

The use of MS is growing, especially with the

development of the more portable and less

costly mass selective detector (MSD).

The MSD and ion trap detector (ITD) may

become more routinely used for pesticide residue analysis as improvements in their computer

software are made and their scan parameters

become more suitable for chromatography.

Both detectors operate on the principle of mass

spectrometry. They differ primarily in the manner in which ions are filtered and in the software that is available for controlling the scan

parameters and data acquisition. Both can be

set to monitor one or more ions during the development of a chromatogram, and both can

take full scans (mass spectra) of chromatographic peaks. Consequently, these detectors

can be used to acquire quantitative and structural data on chromatographic peaks; both are

compatible with capillary columns.

A large and significant difference exists in

the way in which selected ions can be monitored during the chromatography, however.

Only the MSD can be programmed to change

which ions are being monitored during the

chromatogram; this allows the instrument to

be set so that as suspected pesticides elute from

the column, the ions that give the greatest response and are characteristic of the molecule

can be sequentially monitored. The ITD does

not have this capability. Both detectors have

the disadvantage that if nothing is known about

the nature of the sample, they cannot be programmed for selected ion monitoring.

High Performance Liquid

Chromatography Dectectors

The HPLC detectors used for pesticide residue analysis are the UV absorption, fluorometer, conductivity, and electrochemical. A summary of the characteristics of those HPLC

detectors is presented in table 3-4. The fixed

wavelength UV absorbance detector is used frequently for trace analysis of pesticides. Many

pesticides absorb UV light at the wavelength

of mercury discharge (254 nanometers) and can

be detected in very small quantities. Unfortunately, many food coextractives do so as well,

making this detector nearly useless for trace

analysis in foods.

An alternative is the variable wavelength detector, which can be tuned to a wavelength that

is absorbed by the pesticide but not by the food

coextractives. Several successes have been observed using the variable wavelength detector

for “unclean” food extracts, including oxamyl

and methomyl on strawberries (15). A newer

version of the variable wavelength detector, the

photo-diode array detector, is capable of molecular identification for the suspected pesticide

because it is capable of taking a complete absorption spectrum on a chromatographic peak

during the chromatogram. Recent versions of

this detector approach the limits of detection

observed for contemporary variable wavelength

detectors.

The fluorometer is a highly sensitive HPLC

detector for some pesticides. Typically, it is

used for pesticides with aromatic molecular

structures such as alachlor or paraquat. This

detector, however, has limited application to
37

Table 3.4.—Detectors Used In High Performance Liquid Chromatography

Analysis of Pesticide Residues

Full-scale Sensitivity

sensitivity to favorable Temperature

Type of device Units at + 1 noise sample sensitivity

UV Absorption . . . . . . . . . . . . . . . . . . . . . AU’ 0.001 5x10

-

’

0 g/ml Negligible

Fluorometer . . . . . . . . . . . . . . . . . . . . . . . 10 -10 g/ml —

Conductivity ... ... ... ... ... ... ... ..umho2 0.0 5 1 0

-8 g/ml 2 % /

o

c

Amperometric

(Electrochemical . . . . . . . . . . . . . . . . . A3

5 X1O-9 1 0

-10 g/ml 1%/oC

IAU = absorbance units

2Jmho = unit of conductivity; 1mho=ohm-l

3 A = amperes

SOURCE: Office of Technology Assessment, 1988.

the detection of most pesticides—those that do

not fluoresce appreciably. Two ways exist to

avoid this dilemma: labeling the pesticides with

fluorescent molecules before chromatography

by HPLC or by forming postcolumn fluorescent

derivatives (11, 12). Another recent approach

is to form fluorescent molecules from pesticides

by photolyzing them in a photoreactor (7) and

then measuring their fluorescence.

For compounds having photo-ionizable functional groups, the photoconductivity detector

is especially advantageous over UV detectors.

It has been well studied and used by FDA and

other laboratories for residue analysis. The electrochemical detector is also under study for its

potential to improve detection of electroactive

functional groups,

Detection Techniques for Thin Layer

Chromatography

The spots or bands produced after the development of a thin layer plate are detected using

one of several techniques such as visualization

under UV light. Another technique uses reagents to produce colors resulting from chemical reaction that is specific for the pesticide/reagent combination. Amounts of pesticide can

be judged semiquantitatively by comparison

with standards that are developed on the same

plate as the unknowns. An extensive review of

how this technique can be applied to pesticide

residues in foods has been published recently

(17).

A popular color reaction used to visualize and

quantify pesticides separated by TLC is produced by a cholinesterase enzyme-linked chemical reaction (9, 10, 18). Thin layer chromatograms are developed in a tank in the normal

way, removed from the tank and sprayed with

a solution of the cholinesterase enzyme. The

plate is then sprayed with a solution of the colorgenerating reagent; where inhibition of the enzyme by the pesticide occurs, the reagent is not

hydrolyzed, and coloring does not occur in

those areas of the plate occupied by the cholinesterase-inhibiting pesticide. Such an enzyme

inhibition approach was used in the development of a postcolumn detector for the analysis

of carbamate pesticides by HPLC (13). Both

techniques are capable of analyzing nanogram

amounts of insecticide. More recently, there

have been several applications of the enzymelinked Hill reaction for detecting photosynthesis-inhibiting herbicides, such as the triazines,

phenyl ureas, and anilides following TLC separation (4, 5, 6, 14).

CONCLUSION

The techniques currently used in the analy- relatively recent development of SFE and SPE

sis of pesticide residues in food permit precise promises to increase the efficiency of pesticide

and accurate detection and quantification of removal from food material and reduce analytrace levels of hundreds of these chemicals. The sis time. Technological advances in GC column
packing material, composition, wall coatings

and size, and detectors have improved not only

the sensitivity but also the specificity of the analyses performed. The detection and quantification in foods of an increased number of polar

and thermally labile pesticides and metabolizes

have been made possible by the increased use

of HPLC, a technique that has also been improved in recent years. SFC may further enhance the ability to detect this group of pesticides. All of these techniques will continue to

be refined not only as discrete and sequential

steps in analytical method development, but

also as equivalent and parallel steps to increase

CHAPTER 3

1,

2.

3.

4.

5.

6.

7,

8.

9.

Ambrus, A., and Thier, H. P,, “Application of

Multi residue Procedures in Pesticide Residues

Analysis,” Pure and Applied Chemistry 58(7):

1035-1062, July 1986.

Freeman, R. R., and Hayes, M. A., “Column

Considerations When Doing Trace Analysis on

Open Tubular Columns,” J. of Chromatographic

Science 26(4):138-141, April 1988.

Jennings, W., Analytical Gas Chromatography

(New York: Academic Press, Inc., 1987).

Kovac, J., and Henselova, M., “Detection of Triazine Herbicides In Soil By A Hill-Reaction Inhibition Technique After Thin Layer Chromatography, ” J. of Chromatography 133(2):420-

422, March 1977,

Kovac, J., and Henselova, M., “A Rapid Method

for Detection of Hill Reaction Inhibitors,” Photosynthetic 10(3):343-344, 1976.

Lawrence, J, F., “Simple Sensitive and Selective Thin-Layer Chromatographic Technique

For Detecting Some Photosynthesis Inhibiting

Herbicides,” J. Assoc. OfL Anal. Chem. 63(4):

758-761, 1980.

Luchtefeld, R. G., “An HPLC Detection System

For Phenyl Urea Herbicides Using Post-column Photolysis and Chemical Derivatization, ”

J. of Chromatographic Science 23(1 1):516-520,

November 1985.

McNally, M, A. P., and Wheeler, J. R., “Supercritical Fluid Extraction Coupled With Supercritical Fluid Chromatography for the Separation of Sulfonylurea Herbicides and Their

Metabolizes From Complex Matrices,” J. of

Chromatography 435:63-71, 1988.

Mendoza, C. E., Wales, P. J., McLeod, H, A,, et

the number of pesticides that can be determined

in a single sample by a single method (e.g.,

multi-detector systems, e.g., Luke procedure;

see ch. 6).

However, as techniques are improved by

changes in instrument and hardware design,

bringing about more sensitive, selective, and

reproducible devices, their costs usually increase, particularly when automated sample

handling and data manipulation are included.

These additional costs translate into higher

costs to implement contemporary pesticide

methodologies for food.

10

11.

al., “Enzymatic Detection of Ten Organophosphorus Pesticides and Carbaryl on Thin Layer

Chromatograms: An Evaluation of Indoxyl,

Substituted Indoxyl and l-Napthyl Acetates As

Substrates Of Esterases,” Analyst 93(1102):34-

38, January 1968.

Mendoza, C. E., Wales, P, J., McLeod, H, A., et

al., “Thin-Layer Chromatographic-Enzyme Inhibition Procedure To Screen For Organophosphorus Pesticides In Plant Extracts Without

Elaborate Clean Up,” Analyst 93(_1104]:173-

177, March 1968.

Miles, C. J., and Moye, H. A., “Extraction of

Glyphosate Herbicide from Soil and Clay Minerals and Determination of Residues in Soil, ”

J. of Agricultural and Food Chemistry 36(3):

486-491, May/June 1988.

12. Miles, C. J,, Wallace, L. R., and Moye, H. A., “Determination of Glyphosate Herbicide and (Aminomethyl) Phosphoric Acid in Natural Waters by

Liquid Chromatography Using Pre-column

Fluorogenic Labeling with Fluorenylmethyl

chloroformate, ” J. of Assoc. Off. Anal. Chem.

69(3):458-461, May/June 1986.

13. Moye, H. A., and Wade, T. E., “A Fluorometric Enzyme Inhibition Detector For Carbamate

Pesticide Analysis By High Speed Liquid Chromatography,” Analytical Letters 9(10):891-920,

1976.

14. Sackmauerova, M,, and Kovac, J,, “Thin-Layer

Chromatographic Determination of Triazine

and Urea Herbicides in Water by Hill-Reaction

Inhibition Detection Technique, ” Fresenius

Zeitschrift fur Analytische Chemie 292(5):414-

415, 1978.
—.

33

15. Scherer, S. J., Pesticide Research Laboratory, Sherma (eds.) (New York: Academic Press,

University of Florida, Gainesville, FL, personal 1986).

communication, Apr. 12, 1988. 18. Wales, P. J,, McLeod, H. A., and McKinley,

*16. Seiber, J., “Conventional Pesticide Analytical

Methods: How Can They Be Improved?” OTA

commissioned paper, Spring 1988.

17. Sherma, J., “Modern Analytical Techniques, ”

Analytical Methods for Pesticides and Plant 19

Growth Regulators, vol. XIV, G. Zweig and J.

*This reference paper is contained in appendix 13.

W. P., “TLC-enzyme Inhibition Procedure ~o

Detect Some Carbamate Standards and Carbaryl in Food Extracts,” /. Assoc. Off. Anal.

Chem. 51(6):1239-1242, November 1968,

Wright, B. W., Wright, C. W., Gale, R. W,, et al.,

“Analytical Supercritical Fluid Extractions of

Adsorbent Materials, ” Analytical Chemistry

59:38-44, January 1987.
Chapter 4

Immunoassay: An Emerging

Technology
Contents

Page

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

The Immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Strengths and Weaknesses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

Status of Regulatory Use of and Research on Immunoassays . . . . . . . . . . . . . 41

The Potential Role of Immunoassays

Pesticide Residues in fold. . . . . . .

Chapt 4 References ... ... .....C.

Box

4-A. Enzyme-Linked Immunosorbent

in the Detection of

.00.,.. .,.,0.., ,..,,,,. ,,,.,.., . . s 44

.,..,.. . . . . . . . . ,,*,,

B o x

Assay (ELISA). . . . . . .

Figure .

4-1. Artist’s Ccmception of Antibody-Antigen

Tables

Interactions. .

Table

4-1. Immunoamays for Pesticides Under Development by

Regulatory Agencies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

4-2.Comercially Available ELISA Test Kits for Pesticides

.....00 ● ...*, * 45

Page

● *.*.** ● ***** . 39

Page

.,**.*. ● ***** * 37

—

● ✎✎☛✎☛✌ .,00., . 42

*****.. ● ***** * 43
Chapter

Immunoassay: An Emerging Technology

Immunoassay, which use antibodies to detect chemical compounds, are widely used in

clinical chemistry but have not been equally

applied to the analysis of pesticide residues (4).

Yet they seem to have a potentially significant

role in analyzing pesticide residues in food.

Antibodies can be developed to identify single

pesticides or, in some cases, small groups of

similar pesticides. Those immunoassay that

determine groups of pesticides supply data for

the entire group and not the individual pesticides. Immunoassay can also be used to provide quantitative data, similar to that provided

by conventional analytical techniques, or they

may provide qualitative or semiquantitative

data. The latter type can, in some cases, yield

results more quickly than conventional techniques.

U.S. Federal and State agencies do not currently use immunoassay in their pesticide residue regulatory programs on food. However,

FSIS has begun implementing the use of an immunoassay to detect a small group of pesticides,

and Canada’s Department of National Health

and welfare (which regulates pesticide residues

in food) will be training laboratory personnel

in the fall of 1988 in the use of a specific immunoassay to determine one pesticide (1,13).

In higher animals, specialized cells (known

as B lymphocytes) recognize substances foreign

to the body (known as antigens) and respond

by producing antibodies that recognize and

bind to the antigens (figure 4-1). The introduction of a pesticide can stimulate an animal’s

immunological system to develop antibodies

that will recognize and bind to that specific pesticide. These antibodies can be obtained from

the animal’s serum and used for the detection

of the pesticide. However, antibodies are so specific that it is important to decide upon the purpose of the antibody before development begins. For example, for pesticides that are

metabolized quickly, antibodies may need to

be developed for the significant metabolizes

rather than the original pesticide.

Pesticides are usually made up of molecules

too small to induce the production of antibodies. Therefore, pesticides must first be conjugated to a larger carrier molecule, often a proFigure 4-1.-Artist’s Conception of Antibody-Antigen

Interactions

Antibody-antigen interactions result from a precise f it between a surface feature of the antigen and the correspondingly shaped binding sites in the antibody molecules.

SOURCE: Environmental Monitoring Systems Laboratory Las Vegas, Environmental Protection Agency.

3 7
38

tein. Once attached to the carrier molecule, the

pesticide is called a hapten. Where the conjugation occurs will influence the types of antibodies produced. The chemical synthesis of the

hapten-carrier conjugate is generally considered to be the most important factor in obtaining useful antibodies for analytical use, and the

chemistry involved is a major factor in the cost

of immunoassay development (9). The haptencarrier conjugate is then injected into a vertebrate, e.g., a rodent or rabbit, or for large

amounts of antibodies, a sheep or goat. The animal will produce an array of antibodies; some

will bind to the carrier molecule, some to the

hapten-carrier conjugate, and some to the hapten. Only the last of these is useful for developing an immunoassay to detect the pesticide.

These antibodies will be heterogeneous because

different B lymphocytes may produce antibodies that bind to slightly different sites on the

hapten. These antibodies are known as polyclonal because they are produced from a number of different B lymphocyte clones in the animal. They need to be characterized for their

affinity for the hapten (the strength of their binding interaction with the hapten) and their specificity (whether they bind only to the hapten or

to other related chemicals as well).

For most immunoassays, the greater the affinity, then the greater the sensitivity of the analysis (10). The degree of specificity must be

known to determine if the antibody will bind

to, or cross-react with, compounds other than

the hapten. The mixture of antibodies will vary

inside the animal producing them with changes

in the number of each type of B lymphocyte;

it will also vary between each animal immunized.

The changes in the proportion of hapten-specific antibodies and the existence of other antibodies can interfere with the analytical application of a polyclonal-based immunoassay.

By analyzing known concentrations of a pesticide along with the unknown concentrations

in a sample, these variations can be adjusted

for, and successful polyclonal-based immunoassay for pesticides can be developed (10, 11).

The production of monoclinal antibodies can

offer some benefits over polyclonal antibodies,

but some tradeoffs exist. Monoclinal antibodies are produced through a fusion of mouse or

rat B lymphocyte spleen cells with myeloma

tumor cells to produce hybridoma cells, a small

percentage of which will produce the desired

antibody. The spleen is normally taken from

an animal that has first successfully produced

useful polyclonal antibodies. This process takes

a minimum of 3 months before large quantities of antibodies can be produced (21). Hybridomas can live almost indefinitely and can produce an unlimited amount of homogeneous

monoclinal antibodies without the interfering

antibodies that may exist with polyclonal antibodies. And like polyclonal antibodies, hybridomas can be stored in liquid nitrogen and

easily distributed between laboratories.

Monoclinal antibodies, however, are not necessarily the better choice for a specific immunoassay. Polyclonal-based immunoassay may be

adequate for an immunoassay, and in some

cases, they are the more sensitive of the two

(3). But for other pesticides, monoclonals may

be necessary (3). Production of monoclinal antibodies requires more time, labor, equipment,

and training than polyclonal antibodies and can

add 25 percent to development costs (7). New

techniques now under development may reduce

the costs of hybridoma production, however (9).

Polyclonal or monoclinal antibodies are next

incorporated into an immunoassay. Immunoassay for pesticides operate by competitive inhibition, or displacement, in which the antibodies are simultaneously exposed to an unknown

amount of a pesticide in the sample and to a

known quantity of the pesticide separate from

the sample. The more pesticide in the sample,

the fewer antibodies will bind to the latter pesticide (4).

To allow measurement, some sort of tracer

must be attached to either the antibody or the

pesticide. Currently, the most widely used

tracer is an enzyme that will generate an easily measurable color when an additional substance is added. Other tracers include radioisotopes, fluorescent molecules, and magnetic

particles (10). The radioimmunoassay (RIA),

while in some ways more effective than the enzyme immunoassay, currently receives less at-
39

tention for pesticide detection because of the

demands and hazards of working with radioactive substances and because enzyme immunoassay have become increasingly practical (13).

The fluorescent immunoassay, currently used

in clinical applications, potentially may become

as, or more, important for pesticide residue

analysis in food as the enzyme immunoassay.

It can be faster, more sensitive, and more easily automated than the enzyme immunoassay

(9).

To determine the amount of pesticide in a

sample, a standard curve is prepared. Several

different known quantities of the pesticide

(called standards) are separately analyzed with

the immunoassay. A standard curve is prepared

from these results and usually based on the ratio of the amount of pesticide in the standard

to the measurement of the tracer (e.g., the intensity of color produced by an enzyme tracer).

The measurement of the tracer from an assay

of a sample can then be compared against the

standard curve to determine the amount of the

pesticide in the sample (see box 4-A).

Some extraction and possibly cleanup of the

sample may be required before the antibodies

can be used. For some aqueous solutions such

as juices, immunoassay may be applied directly. Immunoassay for some vegetables and

fruits have also been used without a cleanup

step (14). However, cleanup is commonly necPhoto credit: Environmental Monitoring Support Laboratory-Las Vegas,

Environmental Protection Agency

A microprocessor-controlled photometer automatically

reads samples in the 96-well microtiter plate within

2 minutes and provides results in report form.

Box 4-A.—Enzyme-Linked Immunosorbent

Assay (ELISA)

ELISA is a common example of an immunoassay using an enzyme tracer. A test tube or

well in a 96-well plastic microtiter plate is

coated with a known amount of pesticide (conjugated to the carrier molecule) and so the pesticide is immobilized on the surface of the tube

or well.

The sample extract containing an unknown

amount of the same pesticide is added to the

tube or wells. In separate tubes or wells on the

plate, known concentrations of the pesticide

(the standards) are added instead of the sample extract. The antibody that recognizes and

binds to that pesticide is then added. Some of

the antibody binds to the immobilized pesticide and some to the pesticide in the sample

extract or the standards. How much antibody

binds to the immobilized pesticide depends on

how much pesticide is in the extractor standard,

The extract is washed away, and the amount

of antibody bound to the immobilized pesticide will next be measured using the enzyme

tracer. A tracer enzyme may be already attached to the antibody or may be attached by

adding a second antibody (that binds to the

first) conjugated with the enzyme. If the latter is done, then any unbound second antibody

is washed away. A solution of colorless subtrate is added, which will be changed by the

enzyme to a colored product (2 I).

The amount of antibodies bound to the immobilized pesticide is shown by intensity of

the color; the greater the intensity, the less pesticide is in the sample. The intensity of the

color can be measured through the use of a

microspectrophotometer, which may be linked

to a computer with data-analyzing software.

This measurement is then compared against

a standard curve, derived from the standards,

to give the amount of pesticide in the sample.

essary and time-consuming for food containing oil or fat.1

Most immunoassay work has taken place in

academic and regulatory laboratories (14). The

time to develop an immunoassay can vary.

‘For a more detailed description of the technology see ref. 10.
40

polyclonal-based immunoassay generally require 9 months or longer to develop (13), and

monoclinal immunoassay may take a year or

more (21). However, commercial laboratories

having abundant resources and personnel may

be significantly faster than smaller laboratories

in developing an immunoassay, sometimes as

much as 4 to 5 months faster (7).

Another potential application of antibodies

is the biosensor, which theoretically can provide real-time, continuous monitoring of pesticides in a matrix. The biosensor uses biological molecules, such as antibodies, to recognize

and bind to the desired pesticide and a mechanism whereby the binding generates an electrical signal that can be measured and converted to give the concentration of the antigen

(10). Compared to immunoassays, the application of biosensors for the detection of pesticide

residues in food is uncertain.

STRENGTHS AND WEAKNESSES

Immunoassay are particularly suited for polar, water-soluble pesticides and their degradation products that are generally difficult to

analyze using conventional analytical methods.

Because immunoassay can determine biorational pesticides (such as Bacillus thuringiensis), they could be important if use of biological pesticides increases (10). They also can be

significantly faster than certain conventional

methods. Comparisons of quantitative immunoassay with conventional single residue methods using gas or liquid chromatography to analyze specific pesticide/food commodities show

that immunoassay can analyze four to five

times as many samples in a given time period

(15, 16, 17).

The rapid nature of immunoassay is based

on a number of factors. The cleanup step can

be avoided or abbreviated for aqueous samples

(such as juices and milk) and for many fruits

and vegetables. The detection step can be faster

than in conventional methods. For qualitative

and semiquantitative immunoassays, the detection step may take no more than 5 minutes. For

quantitative immunoassays, the use of a 96-well

microtiter plate and plate reader allows detection and quantification of a large number of

samples at one time. Quantitative immunoassay take approximately 4% to 6 hours to perform on food, from sample preparation to detection. Liquids can take significantly less time.

At Health and Welfare Canada, one person can

analyze 12 to 16 fruit and vegetable samples

in triplicate (along with controls) in one day.

This work has been for research, not for regulatory application, and Health and Welfare Canada believes the number of samples could be

tripled for regulatory application (14). Therefore, although the quantitative immunoassay

procedure may take as long as a conventional

method, more samples can be analyzed at one

time.

The use of automation and robotics could further increase the number of samples analyzed.

The principal steps of an ELISA that can be

automated include coating of the wells or tubes

with the immobilized pesticide; washing; addition of antibody, standards, and samples; and

color reading. Systems are available for automating one or more of these steps. For example, unattended, automated spectrophotometers can read 10 to 25 microtiter plates and

record the results in report form. However, because most enzyme immunoassay have long

incubation periods, automation of the entire

procedure by a single unit is not practical yet (9).

In addition, immunoassay can be simpler

to use than conventional techniques, require

less skilled personnel, and require minimal instrumentation time and comparatively inexpensive equipment. Technicians can be trained

within 2 weeks (8, 13). And given that immunoassay can be more portable and simpler to use,

they may be adaptable to field use for food. The

actual costs of an immunoassay used on food

for pesticide analysis versus a conventional

method have not been compared (13). But the

costs of analyzing a sample in general and for

specific nonfood matrices with an immunoas-
41

say are lower than for conventional techniques

(9, 22).

Despite these advantages, the use of immunoassay for monitoring pesticide residues in food

has been constrained by a number of factors.

Immunoassay may not be as sensitive for some

compounds as conventional methods, and they

can have lower levels of reproducibility. Because immunoassay are compound-specific,

they are not suitable for multi residue analysis.

Therefore, while they may analyze more samples in a given time than multiresidue methods, they can detect fewer pesticides.

Characteristics of the food or the pesticide,

in some cases, may also preclude the use of immunoassays. For food samples and pesticides

requiring considerable cleanup work, immunoassay may be no faster than conventional techniques. In addition, immunoassay may not

work well in certain foods. For some pesticides,

e.g., those of very small molecules or having

nonrigid structures, it may not be possible to

develop antibodies. Or if the pesticide has little aqueous volubility, it may not be possible

to use an immunoassay.

Not enough is known about possible crossreactivity of specific antibodies with other

chemicals present in food. Problems caused by

cross-reactivity are a concern but can be controlled if the antibodies are first well characterized and if blank samples and samples with

known concentrations are analyzed at the same

time with the sample in question (4). Crossreactivity can also be a benefit if an immunoassay is needed for a group of similar pesticides.

Another constraint to the use of immunoassay for pesticide residue analysis in food seems

to be the reluctance of some analytical chemists

to explore the potentials of immunoassays. This

is due in part to analytical chemists’ general

unfamiliarity with the biologically based technology. This constraint may be overcome by

validation of the technique and increased training in its use. The speed of doing so will depend on institutional commitment, however.

STATUS OF REGULATORY USE OF AND

RESEARCH ON IMMUNOASSAYS

Antibodies have been developed and reported

for at least 30 pesticides, though few have been

applied to food (for a listing of immunoassay

developed for agrichemicals, see ref. 11). Currently, no government agency has used immunoassay for regulation of pesticide residues in

food, but many are supporting research and development for immunoassay determination of

pesticides, in some cases for matrices other

than food (see table 4-1).

Regulatory agencies’ acceptance of immunoassay vary. Health and Welfare Canada is the

furthest along in the development of immunoassay for pesticide testing in food, Since 1980,

Canada has developed seven immunoassay for

use in food and is currently developing one for

2,4-D. Canada has focused its work on quantitative polyclonal-based ELISAs for polar compounds in non-fatty foods. Canada’s regulatory

laboratories are not yet using immunoassays,

but a planned fall 1988 training workshop on

an ELISA for carbendazim is a first step toward

transferring the technology to the field laboratories (13).

FSIS recently has decided that immunoassay

can have an important role in its regulatory program. This is in part a response to the National

Research Council’s recommendations to test

more samples and to test for more chemicals

using more rapid methods.2 FSIS is now working on implementing a semiquantitative immunoassay for the rapid detection of a group

of five pyrethrin insecticides for regulatory use

in 1989 at its Athens, GA, laboratory. Part of

‘In response to a request from FSIS, the Committee on the

Scientific Basis of the Nation’s Meat and Poultry Inspection Program, Food and Nutrition Board of the National Research Council

prepared in 1985 the report Meat and Poultrjr Inspection: ThP

Scientific Basis of the Nation Program (12), which included

technical recommendations for FSIS’S inspection program.
4 2

Table 4-1 .—lmmunoassays for Pesticides Under Development by Regulatory Agencies

Agency Pesticide

FSIS . . . . . . . . . heptachlor &

heptachlor expoxide

triazines b

FDA . . . . . . . . . paraquat

fenamiphos,

fenamiphos sulfone

and sulfoxide

benomyl, carbendazim, thiophanate

methyl

glyphosate

EPA. . . . . . . . . . paraquat

pentachlorophenol c

atrazine & simazined

CDFA . . . . . . . . molinate

thiobencarb

atrazine & simazine

Health &Welfare

Canada. . . . .2,4-D

Matrix

meat & poultry

meat & poultry

potatoes

oranges

apples

soybeans

soil & water

water

soil

water

water

water & soil

Type of Type of Data

assay antibody a provided

ELlSA M quantitative

ELlSA P qualitative

ELlSA M quantitative

ELlSA M quantitative

ELlSA M quantitative

ELlSA

ELlSA

ELlSA

ELISA

ELlSA

ELISA

ELlSA

ELISA

M

P & M

M

M

M

M

M

P

quantitative

quantitative

quantitative

quantitative

quantitative

quantitative

quantitative

quantitative

Contractor

(if one)

Lawrence Livermore Laboratory

Research Triangle Institute (RTI)

RTI

RTI

RTI

University of California—

Davis & Berkeley

EPA—Las Vegas Iaboratorye

ap = Polyclonal and M = monoclinal.

bFSIS Is evaluating a rapid ELlsA test for tri~]n”s d“@Oped by Immunosystems Inc.

CEpA is “valuating a penta~hlorophenol immunoassay developed by Westinghouse Bio.Analytic Systems COmpany.

dEpA IS d“velopln~ the soil “XtraCtiOn technique for the immunoassay under contract with CDFA.

eThe university of California at Davis is doing the hapten synthesis work and developing polyclonal antibodies. The University of California at Berkeley is developing

the monoclinal antibodies. The EPA Las Vegas laboratory is developing an extraction technique for atrazine and simazine in soil samples.

SOURCE: Office of Technology Assessment, 19SB.

this work is on completing the extraction and

cleanup steps for the immunoassay (l). FSIS

has contracted for the development of other

ELISAS for heptachlor and a number of animal drugs. In addition to contracting for the

development of immunoassays, FSIS also tests

commercially developed test kits and is currently evaluating a commercial qualitative immunoassay for triazine herbicides. FSIS’s use

of immunoassay is made more difficult because

it works primarily with fatty commodities—

meat and poultry—which normally require significant cleanup.

FDA has no current plans to implement the

use of immunoassay for regulatory work. FDA,

however, has a 3-year, approximately $500,000

contract begun in September 1987 for the development of six complete, quantitative immunoassay methods based on monoclinal antibodies (2). FDA is taking a somewhat “wait and

see” attitude on the results of this research before determining the role of immunoassay in

its pesticide regulatory program.

EPA has established a program at its Las

Vegas Environmental Monitoring Systems Laboratory on using immunoassay for the detection of hazardous substances, including pesticides, in the environment. The program tests

commercially available immunoassay as well

as develops immunoassays. EPA does not address food but it has an interagency agreement

with FSIS for the development of antibodies

of common interest,

The California Department of Food and Agriculture (CDFA) has contracted for the development of three immunoassay for use on environmental matrices: soil, surface water, and

groundwater. CDFA has no plans yet to use

these immunoassay on food (19).

Agencies have also taken different approaches

to the development of immunoassay. FDA, for

example, has contracted for the development

of entire immunoassays. EPA has cooperative

agreements with university laboratories to provide hapten work, antibodies, and in some cases

the entire immunoassay. Health and Welfare

Canada has developed its immunoassay inhouse. It seems that enough outside expertise

exists in antibody development for regulatory

agencies to tap using contracts instead of hav-
43

ing to develop the capability to do such work

in-house. Agencies, however, would need some

in-house expertise, at least to identify the types

of antibodies needed, to evaluate the results of

the antibody development, and to adapt the immunoassay for use on food. This last capability would also allow agencies to take advantage of antibodies developed by others for

nonfood matrices.

Because the application of immunoassay to

pesticide monitoring in food is new, a great opportunity exists for agency coordination of research. As noted earlier, EPA and FSIS have

an interagency agreement, and some of CDFA’s

work is done at EPA. But neither FDA nor

Health & Welfare Canada seem to be well linked

with one another nor with the other agencies.

Coordination could be stimulated if agencies

jointly listed which pesticides need improved

methods and then identified those best addressed by immunoassay. In this way, development of antibodies useful to all agencies

could be done without duplication of effort.

Commercial development of immunoassay

for analyzing pesticides is also taking place. A

number of rapid immunoassay tests have been

developed by small private firms (see table 4-2).

Many of these test kits were developed for use

on water and require adaptation to food. Currently, FSIS is the only regulatory agency doing adaption work. Quantitative immunoassay

for pesticides are also being developed privately, but again they are not aimed for use in

regulating pesticide residues in food. Identification by Federal agencies of priority immunoassay needs and communicating these needs

to the private sector might stimulate private development of immunoassay for use in food.

In some cases, private companies have developed immunoassay for internal use, and Federal agencies could investigate the possibility

of obtaining and modifying these immunoasTable 4-2.—Commercially Available ELlSA Test Kits for Pesticides

Claimed limit Water &

Pesticide of detection food matricesb Firm

Paraquat 100 ppb water Environmental Diagnostics Inc.

Burlington, NC

Triazine herbicides (atrazine, simazine 1 ppb water, milk, soup, and lmmunoSystems Inc.

propazine) fruit juices Biddeford, ME

Chlordane-related pesticides (chlordane, hep- 1 ppb water

tachlor dieldrin, aldrin endrin, endosulfan) 200 ppb beer

Benomyl 500 ppb water, orange and grape

juice concentrate

Carbofuran 1 ppb water

25 ppb grape juice

100 ppb rice

2,4-D 1 ppb water

100 ppb beer

Triazine herbicides same as triazines above Westinghouse Bio-Analytic

Systems Co. Rockville, MD

Aldicarb 10 ppb water and watermelon

Carbofuran 10 ppb water

Parathion 10 ppb water and fruit juice

Pentachlorophenol 10 ppb water

aPPb = ~afis per billion

bThe~e food matrices are ones that the firms have tested their immunoassay on. Some Of these immUflOaSSaYS were developed to analYze nonfood matrices and

their modification for use on food may rrof be a prlor~ty of the firm.

SOURCE: Office of Technology Assessment, 1988
44

says for agencies’ use on food (10). For example, a polyclonal-based quantitative immunoassay for cyanazine (an atrazine herbicide) was

developed for soil and water by Shell Oil Company and used to provide data for EPA reregistration of the pesticide. The immunoassay has

a detection limit of 0.5 parts per billion and can

analyze five times as many samples a day as

a conventional method using gas chromatography (18).

As a new technology in the pesticide analysis area, immunoassay require rigorous validation before acceptance by analytical chemists

(21). Validation of immunoassay initially could

be accomplished through comparisons with

established methods, although in time agencies

may need new validation and quality control

protocols to address the unique properties of

immunoassay (3). Standardized methods for

using immunoassay and criteria for evaluating data, a general plan for establishing degree

of cross-reactivity, and minimum quality specifications for the materials (including antibodies used) would all benefit the validation of immunoassay (9).

As in the case of research, coordinating the

validation process for immunoassay among

regulatory agencies could be improved, possibly in conjunction with appropriate professional associations such as the Association of

Official Analytical Chemists (AOAC). Currently,

agencies are conducting validations of immunoassay individually. No official validation studies

of immunoassay for pesticide residue detection in food have been submitted to the AOAC.

Health and Welfare Canada validated each of

its immunoassay by analyzing four to five commodities each with four different concentrations of the pesticide, in duplicate or triplicate,

using conventional methods and the immunoassay on each sample (13). EPA used the same

process of analyzing each sample by both a conventional method and an immunoassay in its

validation of a commercial, quantitative immunoassay for pentachlorophenol. For future

evaluations, EPA will prepare individualized

evaluation studies based on a statistically significant number of samples analyzed through

conventional methods, which will eliminate the

need to analyze every sample by conventional

methods (20).

THE POTENTIAL ROLE OF IMMUNOASSAY IN THE DETECTION OF

PESTICIDE RESIDUES IN FOOD

Immunoassay have a number of potential

regulatory roles. The small number of pesticides

each immunoassay can detect means that immunoassay will complement or improve multiresidue methods (MRMs) rather than replace

them. For example, development of immunoassay could be focused on those polar, moderate-to-high health hazardous pesticides that

MRMs cannot address. Current MRMs might

also be improved by analyzing a sample extract

with conventional techniques as well as immunoassay, thus increasing the number of pesticides that could be detected by the MRMs. Additional work would be required to overcome

possible negative effects of extraction solvents

on the immunoassay, For all uses of immunoassay, conventional methods will be necessary

to confirm violations and ensure that the immunoassay are not giving false negative or

false positive results.

Advances in immunoassay technology may

result in immunoassay being submitted to EPA

during the tolerance-setting process to fulfill

the requirement for an analytical method. EPA

has not formally decided if such a method

would be acceptable, and FDA and FSIS would

need to provide input because the submitted

methods are to be used for regulatory work.

Therefore, EPA’s, FDA’s, and FSIS’s capability to use immunoassay could affect the agencies’ decision to accept them as submitted methods. In a worst case scenario, EPA might be

faced with the submission of analytically
45

acceptable, commercially available methods

whose acceptance might be denied because a

regulatory agency does not have the expertise

or equipment to use them.

Quantitative immunoassay could replace impractical conventional single residue techniques,

increase the number of samples analyzed for

certain pesticides (even those for which practical conventional techniques exist), and increase the number of special surveys for specific

pesticides. Increased automation, including

robotics, of the immunoassay would further

support these activities. Once accepted, quantitative immunoassay may also be used as a

confirmatory single residue technique for analysis by conventional methods.

Semiquantitative or qualitative immunoassay could test large numbers of food samples

rapidly for specific pesticides that need to be

monitored but that have shown few violations

in the past. Thus, more time-consuming and

expensive, conventional quantitative methods

could be reserved for confirming violative samples. Currently, some private sector food manufacturers, such as certain baby food producers,

1.

2 .

3.

4 .

5.

use rapid ELISA tests to ensure that the

products they buy do not have illegal residues

of certain pesticides (5,6), The use of immunoassay in monitoring programs may require some rethinking of objectives because

they would enable a greater number of samples to be analyzed but they do not provide the

quantitative data some agencies require.

The ability of immunoassay to analyze large

numbers of samples would make them useful

when a widespread pesticide residue problem

is suspected in a specific commodity or commodities. The large number of samples that

need to be analyzed in this situation can overwhelm a regulatory laboratory using conventional methods. Such tests could be used to sort

out the violative samples and allow the legal

samples to reach the market more rapidly.

In time, the portability and simplicity of immunoassay, especially the semiquantitative

and qualitative ones, could provide the opportunity to perform testing outside of the laboratory. Issues such as how to address extraction

and cleanup needs, training of the field testers,

and how such analysis would fit into current

regulatory programs would first need to be addressed before field testing was implemented.

CHAPTER 4 REFERENCES

Ashworth, R., Chemistry Division, Food Safety

and Inspection Service, Washington, DC, persona] communication, May 13, 1988.

Clower, M., Center for Food Safety and Applied Nutrition, Food and Drug Administration, Washington, DC, personal communication, July 12, 1988.

Hammock, B., Gee, S., Cheung, P., et al., “Utility of Immunoassay in pesticide Trace Analysis,” Pesticide Science and Biotechnology, R.

Greenhalgh and T. Roberts (eds.) (London:

Blackwell Scientific Publications, 1987).

Hammock, B., and Mumma, R., “Potential of

Immunochemical Technology for Pesticide

Analysis,” Pesticide Analytical Methodology,

American Chemical Society Symposium Series 136, J. Harvey, Jr. and G. Zweig (eds.) (Washington, DC: The American Chemical Society,

1980).

Harvey, R., Beech Nut Nutrition Corporation,

Canajoharie, NY, personal communication,

May 20, 1988.

6. Hether, N., Gerber Products Company, Fremont,

MI, personal communication, May 13, 1988.

7. Hunter, K., Westinghouse Bio-Analytic Systems Company, Rockville, MD, personal communication, July 1, 1988.

8. Hunter, K., Westinghouse Bio-Analytic Systems Company, Rockville, MD, personal communication, Apr. 15, 1988.

9. Karu, A., Hybridoma Facility, College of Natural Resources, University of California-Berkeley,

Berkeley, CA, personal communication, June

28, 1988.

* 10. Mumma, R,, and Hunter, K., “Potential of Immunoassay in Monitoring Pesticide Residues

in Foods, ” OTA commissioned paper, Spring

1988.

*These references are contained in appendix B,
46

11.

12.

130

*14.

15.

16.

Mumma, R., and Brady, J., “Immunological Assays for Agrochemicals,” Pesticide Science

and Biotechnology, R. Greenhalgh and T.

Roberts (eds.) (London: Blackwell Scientific

Publications, 1987).

National Research Council, Food and Nutrition Board, Committee on the Scientific Basis

of the Nation’s Meat and Poultry Inspection

Program, Meat and Poultry Inspection: The

Scientific Basis of the Nation Program (Washington, DC: National Academy Press, 1985).

Newsome, W. H., Food Research Division,

Health and Welfare Canada, Ottawa, Canada,

personal communication, May 6, 1988.

Newsome, W. H., and Graham, G., “Pesticide

Residue Monitoring in Canada,” OTA commissioned paper, Spring 1988.

Newsome, W. H,, “Determination of Iprodione

in Foods by ELISA, ” Pesticide Science and

Biotechnolology, R. Greenhalgh and T. Roberts

(eds.) (London: Blackwell Scientific Publications, 1987).

Newsome, W. H., “An Enzyme-linked Immunosorbent Assay for Metalaxyl in Foods, ” Journal of Agricultural and Food Chemistry 33(3):

528-530, Mav/lune 1985.

17,

18.

19.

20.

21.

22.

Newsome, W. H., and Shields, J. B., “A Radioimmunoassay for Benomyl and Methyl 2-

Benzimidazolecarbamate on Food Crops,”

Journal of Agricultural and Food Chemistry

29(2):220-222, 1981.

Sharp, J., Agricultural Products Department,

E.I. DuPont de Nemours & Co., Wilmington,

DE, personal communication, Aug. 2, 1988,

Stoddard, P., California Department of Food

and Agriculture, Sacramento, CA, personal

communication, Apr. 18, 1988.

Van Emon, J., Environmental Monitoring Systems Laboratory, Environmental Protection

Agency, Las Vegas, NV, personal communication, May 3, 1988.

Vanderlaan, M,, Watkins, B. E., and Stanker,

L., “Environmental Monitoring by Immunoassay,” Environ. Sci. Technol. 22(3):247-254,

March 1988.

Wie, S., and Hammock, B., “Development of

Enzyme-Linked Immunosorbent A&ays for

Residue Analysis of Diflubenzuron and BAY

SIR 8514,” Journal of Agricultural and Food

Chemistry 30:949-957, 1982.

“.

*These references are contained in ar)Dendix ~r -. — B—
Chapter 5

Automation in Today’s Pesticide

Laboratory
CONTENTS

Pago

Individual Component Automation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , ... , 49

The Role of Automation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Multiple Component Automation—Robotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

Robotics in the Pesticide Residue Laboratory . . . . . . . . . . . . . . . . . . . . . . . . . 51

Two Principles for Successful Use of Robotics . . . . . . . . . . . . . . . . . . . . . . . 54

Benefits and Limitations of Robotics in the Analytical Laboratory . . . . . . . 54

Chapter 5 References . . . . . . . . . ..,.0.. .,,*.,,, ● ....*.. ● .*..**, ..,**, , 55

Boxe s

Box ~ Page

5-A. The Ideal, Fully Automated Analytical Laboratory . . . . . . . . . . . . . . . . . . 49

5-B. Zymate’s PyTechnology Robotics System . . . . . . . . . . . . . . . . . . . . . . . . . . 52

-.. -“ .

Figure

5-1. Schematic Drawing of Zymate Robotic System. .,,..,.

Figure

Tables

5-1,

5-2,

Laboratory Unit Operations (LUOs) of Robotic Systems

Comparison of Robot and

Page

● ....*. . . . . . . . 53

Page

..*,.*. . . . . . . .

..,*., ,.,.... .

51

53
Chapter

Automation in Today%

Pesticide Laboratory

INDIVIDUAL COMPONENT AUTOMATION

Automation has greatly increased analytical

productivity of pesticide residue laboratories,

and most such laboratories today use some type

of automated equipment. Computers, for instance, have made the identification and quantification of pesticides easier. Automated geI

permeation chromatography and autoinjection

of samples onto chromatography have allowed

unattended work to take place day and night

and permitted analysts to do additional work.

The Role of Auomation

Despite such advances in automation, the

prospect of designing a fully automated analytical laboratory remains an ideal (box 5-A).

The procedures for analyzing a food sample

are time-consuming, and many steps must still

be done manually. A major percentage of the

total analysis time is spent in preparation, extraction, and cleanup. Food is generally subsampled, cut into manageable pieces if necessary, and subsequently blended with solvent to

extract the pesticide. The sample is then filtered

or centrifuged, and the extract is either partitioned with another solvent or concentrated by

evaporation. An optional cleanup step to isolate the pesticide may be required. Finally, the

sample is injected into a gas or liquid chromatography for analysis.

Automating the sample preparation and extraction steps would generate the greatest time

savings, but these steps are the most difficult

to automate because many types of samples require different preparation (10). Consequently,

improvements in automation have focused primarily on the cleanup and determination stages

of pesticide residues in food analysis.

Several types of automated equipment can

be used in the cleanup step. Gel permeation

Box 5-A.—The Ideal, Fully Automated

Analytical Laboratory

A fully automated laboratory, now existing

only on paper, is one that would automatically

process a sample from its entrance into the

laboratory through the production of a written final report. An automated process of this

type would move the sample through a series

of operations whereby it could be subsampled,

chopped, ground, blended, filtered, centrifuged,

and extracted. The extract then could be evaporated, partitioned, redissolved, diluted, dried,

chemically treated, subsampled and chromatographed, Data from the chromatography would

go to a computer, which would identify the

sample, perform calculations on its abundance,

graph the results, collate it with other data,

and produce a hardcopy. Leftover sample or

sample extracts would automatically be moved

back to a refrigerator, freezer or other proper

storage area, where it would be available for

reanalysis if the computer data did not meet certain quality assurance/quality control standards.

Only very few regulatory laboratories have

experience with robotic automation systems.

Given the current cost and capability of automation instrumentation and technology, it is

not yet possible to automate regulatory laboratories totally.

chromatography (GPC) can be automated (16)

(for a description of gel permeation chromatography see ch. 3); in fact, FDA and FSIS use automated GPC, primarily for fatty foods. Requirements for quick results may pose a problem,

however, because automated GPC processes

only one sample at a time.

Further automation of the cleanup step may

be possible with the recent development of an

49
50

evaporation device that can be connected to

the automated GPC. This evaporation device

replaces the gel permeation solvent with one

more suitable for gas chromatography, concentrates the sample through evaporation, and deposits each sample into a sealed vial, which then

can be injected into a chromatography for analysis. Such a device can process various types

of pesticides with excellent reproducibility and

recoveries (3). At present, FDA has not used

automated evaporation equipment, in part because it does not want to use its capital budget

to replace still functional manual evaporators

and concentrators. FSIS laboratories do, however, use such equipment.

Another automated device for cleaning up

food extracts is the DuPont Autoprep System.

This device, used by some FSIS labs, uses centrifugal force rather than gas pressure or

vacuum, as is done by other devices designed

for this purpose. As many as 12 samples can

be processed at a time, only small volumes for

each wash are required (1 to 5 milliliters), and

the pesticide is effectively concentrated for

analysis by chromatography or other means.

The detection step has also been automated.

Samples to be analyzed using gas or liquid chromatography can be loaded on sample trays holdPhoto credit: Analytica/ Blo-Chemistry Laboratories, Inc.

ABC Laboratories’ GPC/Autovap@ system combines

a gel permeation chromatography module with an

evaporation module to allow automated sample

cleanup and concentration for a maximum of

23 sample extracts.

ing as many as 100 miniature vials and capped

to seal-in volatile organic solvents and pesticides. These trays can be refrigerated to prevent the decomposition of thermally unstable

pesticides. Automated sample injectors, also

known as autosamplers, can then inject the

sample into an automated chromatography for

unattended analysis. Autosamplers have the

added advantage of being more precise in their

volumetric sampling than a chemist, resulting

in higher quality analytical data. Autosamplers,

however, do not appear to be used for the

majority of food samples at regulatory laboratories. In some cases, they are considered

slower and more expensive than hand injection (9).

Automation of the detection step has been

greatly facilitated by computerized data processing. Gas and liquid chromatography are

equipped with computers known as integrators.

Integrators determine the retention time of an

unknown chemical, necessary for its identification, and the quantity of the chemical. The

integrator can then provide this information

in report form. An integrator can be programmed

to identify any specified retention time, allowing easier analysis of a specific pesticide. Mass

spectrometer and infrared detectors are equipped

with computers for sample identification that

can search a library to match a sample to a

known mass spectra. Data processing’s importance is seen as increasing with the development of the laboratory information management system (LIMS). The LIMS goes beyond

recording data; it produces tables that could

be included in reports, it tracks samples, and

it provides an electronic “paper trail” for fulfilling the requirements of “good laboratory

practices.’” In addition, a properly designed

LIMS can be linked with virtually any type of

analytical instrument from any manufacturer

and can be used to collect and interpret data

from it. Pesticide residue laboratories have not

‘These are standards describing the quality of instrumental,

procedural, analytical, and personnel performance prescribed

for laboratories conducting studies that support or are intended

to support applications for research or marketing permits for

(a) pesticide products regulated by the EPA (4o C.F.R. Section

160) and (b) products regulated by the FDA (21 C.F.R. Section 58).
51

yet adopted LIMS because of its early stage of

application and its high cost (4).

Further improvements in analytical methods

are possible through automation, but some constraints exist. Given that much of the automated

equipment including robots has high capital

costs, Federal regulatory laboratories with low

or fluctuating capital budgets may have difficulty purchasing such equipment. Second,

manual procedures may be faster than autoMULTIPLE COMPONENT

Robotics is a special type of automation that

allows mechanical manipulation of an object

in a multitask computer-assisted, and reprogrammable manner (6). In the laboratory, the

robot uses systems technology to allow multiple devices to perform such simple laboratory

operations as weighing, dissolving, diluting, extracting, and so forth. The laboratory robot is

a mechanical extension of a computer that allows it to do physical work as well as to process data.

Laboratory robotics is based on the Laboratory Unit Operations (LUOs) concept. LUOs are

individual processes that can be linked to each

other by hardware and by computer software

to achieve a workable, fully automated analytimated ones on a small scale, although automation may provide other benefits, e.g., reducing

analyst exposure to hazardous solvents. Therefore, decisions to increase the use of automated

equipment must consider the goals of monitoring programs and the moneys available. For example, if increased sample throughput were the

primary goal of a monitoring program, then further advances in automation maybe necessary

before its adoption.

AUTOMATION-ROBOTICS

cal procedure. Table 5-1 explains most of the

LUOs that robotic systems can now perform.

The most popular laboratory robotics system

is produced by the Zymark Corporation and is

a modular system that combines robotics, programmable computers, and peripheral instruments to carry out laboratory procedures (box

5-B). In this system, the robot itself does little

work but simply moves the sample from one

workstation to another where the various operations are performed.

Robotics in the Pesticide Residue

Laboratory

The presence of automated chromatography

in the laboratory now permits their overnight

Table 5.1 .—Laboratory Unit Operations (LUOs) of Robotic Systems

LUO Class Definition Example

Weighing Quantitative measurement of sample mass Direct measurement using a balance

Homogenization Reducing sample particle size and creating a Sonication, homogenization, grinding, etc.

uniform sample

Manipulation Physical handling of laboratory materials Moving test tube from rack to balance, capping,

uncapping

Liquid Handling All physical handling of liquids—reagents and Dispensing reagents, pipetting sample, largesamples volume transfer of liquids

Conditioning Modifying and controlling the sample environment Timing (start and stop), temperature (heat and

control), atmosphere (vacuum or gas blanket),

agitation (mix, stir, vortex, shake)

Measurement Direct measurement of physical properties pH, conductivity, absorbance, fluorescence, etc.

Separation Coarse mechanical and precision separations Filtration, extraction (liquid-liquid, liquid-solid),

centrifugation, precipitation, distillation,

recrystallization, electrophoresis

Control Use of calculation and logical decisions in Adding calculation volume of solvent based on

laboratory procedures sample weight

Data Reduction Conversion of raw analytical data to usable Peak integration, spectrum analysis, molecular

information weight distribution

Documentation Creating records and files for retrieval Notebooks, listings, computers

SOURCE: Zymark Corporation, “Laboratory Robotics Handbook,” Hopkinton, MA, 1988.
52

Box 5-B.—Zymate’s PyTechnoIogy Robotics

System

Zymate’s PyTechnology concept of organizing wedge-shaped modules around the central

robot is shown in fig. 5-1. In the PyTechnology robotics system, there are 48 positions

available, the typical module requiring 2 to 5

positions. Each module is called a PySection

and is available for such LUOs as those in table 5-I. Custom modules can also be obtained

on special order, designed to meet the user’s

specifications. Any PySection is simply locked

into position with wing nuts, and electrical

connection is made at the base of the robot

through premounted contacts.

Control of the robot, connected PySections,

and other peripheral analytical instrumentation is accomplished through the Zymate controller, consisting of a keyboard, disk drive,

and an Easy Lab Controller. The EasyLab Controller houses the central processing unit

(CPU), a memory board, and module card for

each laboratory station connected to the system. A second personal computer can be interfaced with the Easy Lab Controller to allow

simultaneous acquisition of data and user interaction.

Analytical procedures are programmed into

the system via Easy Lab Software. Using this

software, the chemist programs a series of defined tasks using a “top down” approach. This

program has three levels of instructions: the

top-level program, the mid-level program, and

the robot commands. As the chemist proceeds

downward toward the robot commands level,

the instructions to the controller become increasingly detailed, so that the last instruction

might be something like “open fingers. ” In

addition to having all the software available

upon delivery for immediate startup and running “real-world” analytical procedures, the

software also can be custom programmed.

and weekend use; however, many analytica

steps are still done manually by highly skilled

technicians who perform the tasks of weighing, chopping, blending, filtering, partitioning,

and evaporating. If such steps could be done

with robotics, these technicians would be free

Photo credit: Zymark Corporation

The robot acting as an arm and hand, moves the sample

to various modules for different processing steps.

to perform more creative tasks such as data

interpretation and method development.

The use of robotics for routine pesticide residue analysis in foods is just beginning. Chemists

at the Residues and Environmental Chemistry

Section of the Plant Protection Division of

Jealotts Hill Research Station in England have

successfully devised a robotics system to analyze the pyrethroid insecticide Karate in apples.

Portions of apples and pears are carried through

weighing, extraction, partitioning, solid phase

extraction (SPE) cleanup, concentration, and

evaporation steps. Table 5-2 compares recovery data for apples and pears by a robot and

by a human. The robot gave more consistent

recoveries for all samples studied.

A robotic system for the determination of the

herbicide tridiphane in rat chow has been developed as part of a toxicology study on that

chemical (7). Recoveries, however, were generally lower using robotics (86.5 percent recov-
53

Figure 5-1.–schematic Drawing of Zymate Robotic System

UV/Vis

spectrophotometer a

\

General

P

(

purpose hand 4

dissolve L

SOURCE” Zymark Corporation, “Lalmratory RobotIcs Handbook,” tlopkmton, MA, 19SS

Table 5-2.—Comparison of Robot and Human

Generated Data

Robot Human

Internal standard Residue Internal standard Residue

Sample no. recovery (%) (mg/kg) recovery (%) (mg/kg )

1 83 0.10 119 0.10

2 84 0.11 127 0.12

3 83 0.09 111 0.07

4 81 0.08 127 0.07

5 88 0.08 127 0.09

SOURCE I Laws and R Jones, “Generic Sample Preparallon System for Automahon of Pestlclde

Analysls, Advances m Laboratory Auforrraf/c Itobot/cs VOI 4 (Hopklnton MA Zymark

Corp 1984)

cry) than by the manual method ($93.0 percent

recovery), and 4% hours were required to process 10 samples by robotics compared with 3%

hours for the manual procedure. On the other

hand, the robot can work 24-hour days, whereas

the technician normally works only 8. Furthermore, robotic recoveries were more consistent.

A robotic system has been used to isolate a

synthetic pyrethroid insecticide from extracts

of soil, sediment, fish, and mussel at levels as

low as 1 part per billion. Small SPE cartridges

packed with Florisil were used to accomplish

this, and observed recoveries averaged 85 percent or more for these sample types (I),

Neither FDA nor FSIS uses or is testing the

use of robots for analyzing pesticide residues

in food, although both agencies are monitoring advances in robotic technology. Health and

Welfare Canada is evaluating two robots in its

laboratories: one to carry out the liquid-liquid

partition step in the Luke method and the other

to carry out the extraction and cleanup steps

on milk samples undergoing a multiresidue test

for organochlorines (12). Early results show the

latter robot to be equivalent in accuracy to man-
54

ual preparation while doubling the weekly output of samples (12).

Two PrincipIes for Successful

Use of Robotics

In a recent survey of numerous firms that

have installed four or more robotics systems,

two principles were mentioned that many felt

were necessary for successful incorporation of

robotics in a laboratory (8):

• A single motivated and well-qualified person must be given the responsibility of seeing that a system is installed and put into

operation in a productive way. That person should be given enough resources and

time so that his or her efforts are not diluted

with other responsibilities. It may be necessary to hire a chemist with some electronics training or experience, since robotics relies on electronic and computer

technologies. Analysts could also be retrained through on-the-job short instruction that would allow persons to improve

their understanding of how things are done

within the framework of the modern robotics system.

● The selection of an initial application or

part of a complex application should have

well-understood chemistries so that rapid

startup with quickly measurable productivity is realized.

Benefits and Limitations of Robotics

in the Analytical Laboratory

The benefits of using robotics in a laboratory

include improved test precision, morale, worker

safety, and “product” quality (2). Robotics in

a regulatory laboratory doing pesticide residue

analyses or method development has other advantages as well. It provides exacting timing

and uniform sample handling, which ensure

precision and accuracy (5). Analytical methods

are transportable from laboratory to laboratory,

since they are stored on computer diskette and

executed by instrumentation that is identical

wherever they are implemented (14). Moreover,

an electronic “paper trail” is left on the computer for all analytical operations performed

on a sample (6, 14). (See Kropscott et al. in appendix B for additional discussion.)

Currently, attention is being focused on designing robots for methods development and

the subsequent method optimization. This

would lead the way toward a robot specifically

designed for pesticide regulatory laboratories.

One common mistake made by those attempting to use robotics in the laboratory is to assume that robots are designed to simulate humans. Robots have a great deal of difficulty with

some operations because their parts simply do

not have the degree of freedom that a human

hand does, for instance. They also do not move

as fast nor do they have the load-carrying capacity of a human. For example, robots have

trouble moving and processing large fruits or

vegetables. They are unable to manipulate some

laboratory glassware that is currently in vogue,

such as large separator funnels or evaporative devices. They are better able to manipulate small tubes, pipettes, flasks, and similar

containers.

Before robotics can be used in existing MRMs

such as the Luke and the Mills-Onley-Gaither

procedures, smaller samples, smaller amounts

of solvent, and more sophisticated evaporative

techniques must be demonstrated to be effective. when necessary, the newer cleanup techniques that reduce sample size requirements,

such as SPE cartridges, need to be evaluated.

Innumerable successes in the drug and petrochemical industries have demonstrated that

great gains can be made in productivity when

procedures can be miniaturized.

Robots have significant costs similar to costs

of conventional computer systems—estimates

range from $60,000 to $120,000 for the purchase

of the robot and the renovation of the laboratory (5, 15). In addition to the cost, significant

time must be given to adapting the robot to the

needs of the laboratory and to familiarizing laboratory personnel with its use. Older model

robots had startup times of 3 to 6 months (15),

and the Health and Welfare Canada robot now

doing milk samples took about a year to set up

(11). However, startup times should decrease
55

dramatically with improvements in the robots

such as pre-programming and with increased

familiarity with their use (13). Lastly, robots are

subject to mechanical and electrical breakdowns and require a continuous power supply.

Robotics then should not be viewed as a cureall for those regulatory agencies now inundated

with food samples, but rather as a supplement

to conventional manual techniques now being

used. It is expensive to acquire, requires a new

way of thinking to use it effectively, and suffers

from the limitations listed above. On the other

hand, it can measurably improve the overall

operation of the analytical laboratory.

CHAPTER 5 REFERENCES

1.

2.

3.

4.

5.

*6.

7.

Akkari, K. H., “Robotic Residue Sample Purification by Solid-Phase Extraction, ” Fifth international Symposium on Laboratory Robotics,

Boston, MA, 1987, Abstr. 1n: Advances in Laboratory Automation Robotics, vol. 4 (Hopkinton, MA: Zymark Corp., 1988).

Curley, J. E., “Making Robots Fly,” Advances

in Laboratory Automation Robotics, vol. 2

(Hopkinton, MA: Zymark Corp., 1985).

Hopper, M. L., and Griffitt, K. R., “Evaluation

of An Automated Gel Permeation Cleanup and

Evaporation System for Determining Pesticide

Residues in Fatty Samples,” J. of Assoc. Off.

Anal. Chem. 70(4):724-726, JulylAugust 1987.

Joe, T., Chemistry Laboratory Services, California Department of Food and Agriculture,

Sacramento, CA, personal communication,

June 3, 1988.

Kropscott, B., Dow Chemical Company, Midland, MI, comment at the OTA workshop

“Technologies to Detect Pesticide Residues in

Food, ” Mar. 15, 1988.

Kropscott, B. E,, Peck, C. N., and Lento, H. G.,

“The Role of Robotic Automation in the Laboratory, ” OTA commissioned paper, Spring

1988.

Kropscott, B. E., and Dittenhafer, M. L., “Automated Feed Analysis—an Application for Tox-

*These reference papers are contained in appendix B.

8.

9,

10,

11,

*12,

13.

14,

15,

16,

icology, ” Advances in Laboratory Automation

Robotics, vol. 1 (Hopkinton, MA: Zymark

Corp., 1984).

Laboratory Robotics Handbook (Hopkinton,

MA: Zymark Corp., 1988),

Luke, M., FDA Pesticide Laboratory, Los Angeles, CA, personal communication, Apr. 26,

1988.

McCullough, F., ABC Laboratories, Columbia,

MO, personal communication, May 3, 1988.

Newsome, W. H., Health Protection Branch,

Health and Welfare Canada, personal communication, May 6, 1988.

Newsome, W. H., and Graham, G. F., “Pesticide

Residue Monitoring in Canada,” OTA commissioned paper, Spring 1988.

Paul, A., Zymark Corporation, Hopkinton,

MA, comment at the OTA workshop “Technologies to Detect Pesticide Residues in Food, ”

Mar. 15, 1988.

Paul, A,, Zymark Corporation, Hopkinton,

MA, personal communication, May 10, 1988.

Peck, C., Dow Chemical Company, Midland,

MI, comment at the OTA workshop “Technologies to Detect Pesticide Residues in Food, ”

Mar. 15, 1988.

Tindle, R. C., and Stallings, D, L,, “Apparatus

for Automated Gel Permeation Cleanup for Pesticide Residue Analysis,” Analytical chem

istry 44(11):1768-1774, September 1972.
Chapter 6

Pesticide Analytical Methods
Content s

Introduction ● **** **. **8*.*****.*,.*.*..**.*,,,*.,*** .**,

Types of Methods   ** ** ***. *o** **** Oo. n *e.***.,,.*****

Multiresidue Methods ● **,6..*****,*.,*,,***, .

Single Residue Methods . . . . . . . . . . . . .

. *,*******

. *********.**,,,**,

Semiquantitative and Qualitative Methods ,0 . . 0 .,, .,,,...0

Current Needs in Methods Development .., .....,.,, . . . . . . .

Cost Considerations of Methods Development Research . . . . . .

Needs for Adoption and USe of Methods ● *.******.,..,***,*

Chapter 6 References ● **, .,*. o*.,*,,,...,.**,*,.***,*,,, ,

Barns

Box

6-A. The Concepts of Screening and Rapid Testing ., , . . . . . . .

6-B. Ongoing Challenges to Methods Development: Metabolizes

New Pesticides. ~. . . . . . . . . . . . . . . . . . .;. . . . . . . . .

Figure

6-1. Decision

Methods

Tree for Testing Pesticides Through FDA

● Q.*. * * s * . . . * * * * *, * * *,, *, * * * ***,....,

6-1. Multiresidue

Table

Methods Routinely Used by

● ☛✌✎✌✎ ☛

Page

● ● ● ● ● ● * . ● ● ● 59

● ● * ● * ● * e * * ● 59

. **,******, 59

● **4*.*,,** 61

. . . . . . . . . . . 61

● ***,,**,* * 64

. . . . . . . . . . . 69

● @ ● . ● ● ● ● ● @ $ 69

8 *******,,, 71

and

., *...**.. , 68

Multiresidue

● ☛✌✎ ☛ ✎ ✎ ✎ ✎ ✎ ✎ ✎ ✎ ✎ ✎ ✎ ✎ ✎ 62

Page

FDA, USDA, and CDFA . . . . 6 0

.
Chapter

Pesticide Analytical Methods

INTRODUCTION

The regulatory responsibilities of FDA and

FSIS influence the type of methods these agencies use to monitor pesticide residues in food.

Methods must provide results that are costeffective, timely, reliable, and verifiable. The

agencies also need methods that can identify

as many pesticides as possible in a range of food

commodities because they are charged with

monitoring all foods for all pesticides. In addition, these methods should use instruments,

associated hardware, and reagents that are

readily available in the regulatory laboratory

or are commercially available and inexpensive.

Regulatory agencies need methods that can

give reliable results rapidly—within 24 hours—

if violative products are to be kept from reaching the market, Neither FDA nor FSIS has the

authority to detain commodities routinely monitored for pesticide residues, but both agencies

can detain imports suspected of illegal residues

and FSIS can detain suspected domestic meat

until the results of analytical testing are received.

Methods must also be able to detect pesticides

at, and often below, tolerance levels. They must

endure interfering compounds such as other

pesticides, drugs, and naturally occurring chemicals and be insensitive to such environmental

TYPES OF

Muitiresidue Methods (MRMs)

MRMs come closest to meeting the method

needs of the regulatory agencies. They are designed to identify and quantify a number of pesticides and their toxicologically significant

metabolizes simultaneously in a range of foods.

Their usefulness is based on a combination of

three factors:

variations as humidity, temperature, and solvent purity. Chemists with varying levels of

training and expertise must be able to use them.

There also should be some other means of confirming that a method is accurate.

EPA provides guidance for methods as part

of the tolerance-setting process that involves

many of these points. According to its Subdivision O Guidelines, submitted methods should

1) take 24 hours, 2) require readily available

equipment or reagents, 3) identify the residue

in the presence of other residues, and 4) detect

the residue at or below the tolerance. EPA’s

guidelines do not include an emphasis on multiresidue methods (MRMs) or the submission

of a confirmatory method.

FSIS also has criteria for methods suitable

for its regulatory use: 1) methods must take no

more than 2 to 4 hours of analytical time per

sample, 2) they must have a minimum proficiency level at or below the tolerance, 3) there

must be a quality assurance plan developed for

the method, and 4) the method must be successfully validated through an interlaboratory study

(6).

FDA does not have a formal listing of guidelines for its methods, but it uses many of the

same criteria in evaluating them (13).

● determining abroad spectrum of pesticides

and their toxicologically significant metabolizes in an array of food,

● being sensitive, precise, and accurate enough

to be useful for regulatory purposes and

acceptable to the scientific community,

● being economical or at least affordable for

those laboratories using them.

59
60

No single method can optimize each of these

three factors; as a result, the MRMs used are

a compromise of these elements (see ref. 20 for

further discussion of this point).

MRMs have two other advantages. An MRM

may be able to detect, but not measure, a particular pesticide or metabolize in food. The

MRM, in such cases, signals the presence of

the compound, which can then be analyzed

with a single residue method (SRM) (16).

Second, MRMs record the presence of unidentified chemicals, known as an unidentified analytical response (UAR). Once observed, the

chemical’s identity can be determined by matching its result to a known chemical with a similar chromatographic result or by other techniques such as mass spectrometry. In this way,

MRMs can identify the presence of possibly

hazardous chemicals that were not expected

to be residues in food and might have been overlooked. For example, polychlorinated biphenyls

(PCBs) were discovered in meat and animal feed

after appearing as UARs on the chromatograms

of samples analyzed for the chlorinated hydrocarbon pesticides.

MRMs contain the steps of preparation, extraction, cleanup, chromatographic separation,

and detection (as described in chapter 3). All

MRMs used today in the United States are

based upon either gas chromatography (GC) or

high performance liquid chromatography (HPLC)

as the determinative step. of the 10 MRMs routinely used by FDA and USDA, 8 rely on GC

as the determinative step (see table 6-l). Thin

layer chromatography is still used by several

agencies in Europe, but it has lost favor in this

country because of its semiquantitative nature

(1)0

The FDA and USDA have geared much of

their pesticide methods research to developing

Table 6-1.–Multiresidue Methods Routinely Used by FDA, USDA, and CDFA

Food type Pesticide groups

Agency Method’ analyzed detected

FDA GC-multiple detectors

(Luke method)

GC-multiple detectors

(Mills method)

GC-multiple detectors

(MOG method)

GC-multiple detectors

(Storherr method)

HPLC-fluorescence

(Krause method)

USDA-FSIS GC-ECD

GC-ECD

(western method)

GC-NPD

(eastern method)

HPLC-fluorescence

USDA-AMS 2 GC-ECD

CDFA3 GC-ECD

GC-NPD or FPD

HPLC-fluorescence

nonfatty

fatty

nonfatty

nonfatty

non fatty

fat

liver and fat

liver

liver

fatty

(raw egg products)

nonfatty

non fatty

nonfatty

organochlorines

organophosphates

organonitrogens

organochlorines

organophosphates

organochlorines

organophosphates

organophosphates

N-methyl carbamates

chlorinated hydrocarbons

chlorinated organophosphates

organophosphates

carbamates

chlorinated hydrocarbons

organochlorines

organophosphates

carbamates

I Methods are identified by the combination of the instruments used for chromatographic Separation and detection. Abbreviations for the these instruments are as follows:

GC: gas chromatography

HPLC: high performance liquid chromatography

ECD: electron capture detector

NPD: nitrogen-phosphorus detector

FPD: flame photometric detector

In some cases, a method may also have a name and these are noted in parentheses.

2AMS: The Agricultural Marketing Service

3CDFA: The California Department of Food and Agriculture

SOURCE Office of Technology Assessment, 1988
61

MRMs over the years. FDA’s MRMs appear in

Volume I of the Pesticide Analytical Manual

(PAM I), and they are considered of high quality and capable of providing data that will withstand challenge during court litigation (for details on the development of FDA’s MRMs see

ref. 19).

The primary weakness of existing MRMs is

that they cannot detect every pesticide. For example, of the 316 pesticides with tolerances,

only 163 of them could be analyzed with FDA’s

five routinely used MRMs. A second weakness

is that some MRMs require a great deal of time

to perform, thereby reducing the number of

samples analyzed and the speed of analysis. For

example, certain foods, such as those with high

concentrations of fats and oils, are difficult to

analyze in a timely manner.

Single Residue Methods (SRMs)

A large number of methods exist that are designed to analyze a single pesticide and, in

many instances, its metabolizes or degradation

products. Although less efficient than MRMs,

the use of SRMs is necessary to monitor those

pesticides, including a number of high health

hazard ones, that cannot be detected by MRMs.

SRMs depend on a number of different techniques and vary widely in terms of reliability,

efficiency, throughput (samples per day), degree of validation, and practicality for regulatory use. SRMs are primarily developed by the

private sector for submission to EPA as part

of the tolerance-setting process, Therefore, a

method exists for every pesticide with a tolerance, although methods for some pesticides

(primarily the older ones) may not be effective.

Most SRMs, like MRMs, are based on GC

using the full array of element-specific detectors. Volume 11 of the Pesticide Analytical Manual (PAM II) consists solely of SRMs, both those

that have undergone EPA review and possibly

EPA laboratory evaluation, and those that have

appeared in a peer-reviewed journal of high

quality (these methods are normally similar to

ones approved by EPA but adapted for other

commodities) (10). In PAM II, those methods

reviewed by EPA are listed with Roman numerals

and those not reviewed are lettered.

SRMs are not considered adequate for routine monitoring by the regulatory agencies, although FDA uses them. To monitor one pesticide with an SRM is considered inefficient

when an MRM can measure many pesticides

using the same resources. In addition, SRMs

vary widely even for chemicals of the same

class, so a laboratory needs a wide array of

glassware, evaporative devices, chromatography,

and detectors to use the SRMs available. There

is also dissatisfaction with the performance of

some SRMs (24). Some chemists feel they are

better served sometimes by 1) going to the scientific literature for methods, 2) borrowing

methods from State laboratories, or 3) going

directly to the registrant for the newest method.

Others feel it is better to develop their own

methods or adapt existing methods developed

for pesticides of similar structure. SRMs are

also not as capable of identifying UARs as

MRMs.

In an attempt to reduce the need to use SRMs,

EPA now requires that all pesticides requiring

a new tolerance be evaluated to see if they can

be detected by FDA and USDA MRMs. Only

FDA has developed the testing protocols to support such testing. FDA has also devised a “decision tree, ” showing the order in which the

FDA MRMs should be tested using the new pesticide to minimize research time (figure 6-1),

The results confirm or deny whether that particular pesticide can be recovered through one

of the MRMs. It has not yet been decided

whether the EPA will still require development

and submission of an SRM if the pesticide can

be analyzed by an MRM (23).

Semiquantitative and Qualitative

Methods

Semiquantitative and qualitative methods

range widely in their ability to quantify the

chemical present in a sample. Semiquantitative

methods indicate the range of pesticide residue concentration in a sample; qualitative methods show whether or not a particular pesticide

exists above some predetermined concentra-
62

Figure 601.– Decision Tree for Testing Pesticides Through FDA Multiresidue Methods

Part A

Does the compound have an N-methyl carbamate structure?

Yes

7 ° \

Is the compound an acid? Test per protocol I v A L

No

o

Yes

/-, , ,

\

Test per Protocol V*

Test through GC

portion of Protocol Ill

I

Does it chromatography

on GLC systems?

Yes

\

Does it chromatography on at least

one column within a reasonable time

(.3 < rrt< 5) at standard conditions?

Go to part B for

further directions

tion. In this way they differ from the majority

of conventional MRMs and SRMs, which fully

quantify the amount of pesticide in a sample.

(See also box 6-A.)

The benefits of these methods maybe their

low cost, speed, or ease of use. These benefits

can contribute to an increase in the number

of samples that could be analyzed, although

tradeoffs may exist in the number of pesticides

that can be analyzed. Currently, neither FDA

nor FSIS is using these methods for pesticide

Part B

For further study of compounds producing reasonable GC

peaks (Perform recovery studies using adjusted GC conditions

if necessary

Is the product a nonfatty (== 2% fat) food?

>

Yes

\

Test per Protocol Ill

\

SOURCE: Food and Drug Adminm.tratlon, Division of Contaminants ChemWy,

March 19SS

monitoring, although both agencies are conducting ongoing research. Similarly, EPA is

conducting research on the use of these methods on nonfood matrices. Given that the majority of these methods have been developed for

pesticides in nonfood matrices, significant

adaptation research may be necessary for their

use on foods.

Semiquantitative and qualitative methods

make use of such technologies as thin layer

chromatography (TLC), enzyme inhibition, and

immunoassay, These three technologies can be

moved from the laboratory into the field without losing their ability to detect pesticides. And

because sophisticated instrumentation is not

required, they are relatively inexpensive compared to quantitative methods.
63

Box 6-A.—The Concepts of Screening and Rapid Testing

Screening and rapid testing are two terms commonly used when discussing improvements in

methods that will determine in a short time whether or not a pesticide or group of pesticides is present

in food. The use of these terms often is confusing because they have different meanings to different

people, and the confusion can be compounded when the two are used together, i.e., “rapid screen. ”

The term screening, in general, can be applied to two different types of methods. The distinction

between the two methods depends on what is being screened--either a large number of pesticides

or a large number of samples.

First, screening can mean a method that can detect a large number of pesticides in a sample,

that is, the method screens for pesticides. The multiresidue methods (MRMs) used by FDA and USDA

are screening methods under this definition. These “MRM screens” analyze for a large number of

pesticides at one time and therefore are the most cost-efficient approach when data on pesticide application are lacking or when a number of pesticides are known to have been used. Also, they can uncover the presence of residues not expected to be in the food. This type of screening method may

also be labeled “rapid test” for a number of reasons. These MRMs are faster than single residue methods (SRMs) because they can analyze for more pesticides in a given time period. Some MRMs are

considered rapid because they are relatively faster than other MRMs. For example, the Luke MRM

used at FDA’s Los Angeles laboratory can detect some 200 pesticides and metabolizes in 30 samples

each day and therefore is considered a rapid screen.

Second, screening is also used to mean a method that can analyze a large number of samples

often for one pesticide or a small group of pesticides in a relatively short period of time, that is, the

method screens samples. This type of screening method supports efficient identification of violative

samples when a known pesticide/commodity problem exists or where a pesticide/commodity combination is known to have a low violation rate. When a method is used in this manner, the speed of

analysis in terms of the number of samples that can be analyzed per unit time is emphasized and,

in this context, it would then be considered a “rapid screen. ” Application of such a rapid screen

to a large number of food samples thought to contain violative samples would allow nonviolative

food to reach the market more quickly and reduce the number of samples that need to be analyzed

by more time-consuming and expensive conventional methods. Those samples with positive results

would be analyzed by a conventional method.

This type of screening uses technologies that are less expensive and more rapid to use than conventional methods, such as thin layer chromatography, immunoassay, and enzyme inhibition. The

lower cost of these techniques stems from their relative speed of analysis and use of less expensive

and more simple equipment. These techniques often are called “rapid tests, ” because of their speed. ’

The tradeoffs of using these techniques are noted in chapters 4 and 6. Neither FDA nor FSIS uses

such screening methods for pesticide monitoring in food, although FSIS is actively researching its use.

IFor more detail on this type of screening, see Ellis 1988 in appendix B.

A drawback of semiquantitative methods is TLC is used sometimes in Europe for regulathat they do not provide the degree of accuracy

necessary for enforcement action, e.g., for use

in a court of law. Violations found by a semiquantitative method would have to be verified

by a quantitative analytical method—or maybe

two. And with the possible exception of thin

layer chromatography, none of the semiquantitative techniques provide data that can be used

to address UARs.

tory purposes (see ref. 1 for a bibliography of

TLC applications). Thin layer chromatographybased methods have the advantage of an ability to analyze several pesticides simultaneously.

As many as 20 pesticides can be tested at once

if chromatographic conditions are properly

chosen. TLC has been used successfully by FSIS

to analyze the drug sulfamethazine in animal

tissues; field use by inspectors relatively un-
64

skilled in analytical chemistry was also successful. An attempt to use TLC for analysis of chlorinated hydrocarbons in animal tissues has

been unsuccessful, however, because of problems in achieving the desired sensitivity and

an overly complicated sample extraction procedure for nonlaboratory use (3). Several potentially useful TLC methods are described in

chapter 4 of PAM I. They are carryovers from

early work at FDA and USDA and require sample cleanup by conventional Florisil or alumina

columns. Both FDA and FSIS have ongoing research on TLC applications.

Enzyme inhibition-based color reactions are

a means of making the spots and bands of pesticide residues on thin layer chromatographic

plates visible in order to measure the pesticide

residue either visually or with instruments.

Such techniques have been developed for cholinesterase-inhibiting insecticides and photosynthesis-inhibiting herbicides.

In addition to working with TLC, enzyme inhibition may also be used for a “stand alone”

test kit. Currently, one such qualitative kit is

commercially available for the detection of

cholinesterase-inhibiting insecticides (organophosphates and carbamates). The kit is inexpensive and can detect a large number of pesticides in concentrations of parts per million.

The kit has been privately used for analyzing

food extracts and for analyzing water used to

wash skins of fruits and vegetables for pesticide residues (9). This type of kit is not specific

unless information about the history of the sample is available. For example, it will give a positive response for a large number of compounds

without being able to identify the specific compounds. This type of kit also may suffer from

interferences produced by extraction solvents.

Immunoassay have been developed for semiquantitative and qualitative tests, although

much immunoassay research has focused on

quantitative assays (17). If needed, quantitative

immunoassay based on color reactions could

be adapted to semiquantitative assays with

visual interpretation of the results. Such tests

could then be more easily used outside the laboratory. Several qualitative tests are commercially available. Immunoassay-based methods

have the advantage of speed since many tests

can be performed simultaneously and some

analyses take less than a few minutes if extractions are not necessary. They also have the

advantage of being extremely sensitive, detecting some pesticides far below their tolerances,

and they are usually specific, although sometimes cross-reactions occur that give false positives (17). A drawback is that immunoassay

provide analyses for individual or small groups

of pesticides.

Current Needs in Methods

Development

improving Existing

Analytical Methods

Considerable time and resources have already

been invested in developing analytical methods. Rather than devoting resources exclusively

to developing new analytical methods, existing methods also can be improved through

changes in technologies to reduce analysis time

and to increase the number of pesticides that

can be analyzed. For instance, improvements

could be made in the following ways:

1.

2,

3.

4.

5.

6.

7.

8.

simplifying cleanup

improving extractions with supercritical

fluid extraction (SFE)

miniaturization with solid phase extraction

(SPE)

capillary columns

increased use of high performance liquid

chromatography [HPLC)

use of immunoassay as a detection technique

increased automation

mass selective detection (MSD)

(1)Simplifying cleanup. Simplifying a method

by eliminating sample manipulation in the

cleanup step would shorten analysis time, eliminate opportunities for pesticide loss, reduce

solvent and consumables usage, and reduce

overall analytical costs.

Two FDA MRMs and the three used by the

California Department of Food and Agriculture

(CDFA) use food extracts that have not undergone any type of sample cleanup. Approximately 80 percent of the food samples analyzed +
65

by FDA are examined with the Luke method,

and CDFA conducts more analyses with its own

three methods than any other State. The trend

toward less extensive sample cleanup in these

methods has been a result of improving capabilities of element-specific detectors (NPD, FDP,

ECD, and Hall).

As sample cleanup is reduced or even eliminated, increased stress is placed on the determinative step. As a result, the chromatographic

separation begins to suffer from the presence

of large amounts of sample coextractives. Such

coextractives may produce a loss of resolution

of pesticides in the sample, a loss of pesticide

on the chromatographic column, and fouling

of the detector. For these reasons, the chemist

must weigh the need to shorten analysis time

with the instrumentation “down time, ” that is,

time required to clean, repair, and regenerate

the instrument to its original operating specifications.

However, because reduction of cleanup steps

pays high returns in time saved for a typical

analysis by reducing analytical costs and increasing sample throughput, efforts should be

made to explore it fully.

(z) Improving extractions with SFE. As more

efficient hardware (particularly miniaturized

valves, pumps, ovens, and refrigeration devices)

becomes available for SFE, the technique may

become more practical for extracting pesticides

in foods, possibly in the field, e.g., the slaughterhouse. SFE can be coupled to capillary column gas chromatography or supercritical fluid

chromatography (see ch. 3) to provide an online extraction/determination, although validated methods have yet to be developed using

this approach. Since extraction time can be

shortened, then selectivity can be gained by

leaving potential interferences behind and thermally unstable chemicals can be dealt with. The

technique has become attractive for consideration in the future. It may ultimately shorten

analysis time while expanding the array of pesticides and metabolizes that can be extracted,

Carbon dioxide, a relatively inert gas, has

been used as an SF for the extraction of many

types of organic compounds. Straight chain

hydrocarbons have been selectively extracted

from other chemicals present (8). More than

85 percent of such hydrocarbons were extracted

in 5 minutes. Extractions can be even more efficient and faster as well as applicable to more

polar chemicals by modifying the carbon dioxide with small amounts of polar organics.

(3) Miniaturization with SPE. An opportunity

to reduce analysis time, solvent consumption,

and overall costs might be through use of miniaturization (20). The philosophy of present miniaturization focuses on the use of small solid

phase extractions (SPEs). These cartridges are

now commercially available, are inexpensive

($2 to $3 each), and are disposable. Use of SPEs

has not been demonstrated yet for MRMs, although they have been successfully used in

SRMs (for such pesticides as aldicarb and paraquat). Problems associated with larger adsorption columns, such as the Florisil columns, may

still exist with SPEs in MRMs. For example,

pesticides may not exit the SPE in distinct

groups but may instead be scattered among several fractions. In addition, there still may be

a problem of pesticide loss on these extraction

columns, depending upon the elution conditions and the pesticide under analysis. Associated with miniaturization are the problems of

taking a truly representative sample, so that

analytical results will reflect the average concentration of the pesticide in the food (2).

Miniaturization of MRMs might assist in

adapting robotics to MRMs (see ch. 5). Present

robotic modules handle samples of 1 to 10

grams better than heavier ones of 25 to 1 0 0

grams, like those used for conventional MRMs.

Similarly, robots dispense and manipulate 5 to

25 milliliters of solvents more easily than the

100 to 250 milliliters typically used in conventional MRMs (12).

Other spinoffs of miniaturization might be

that sample preparation could be done in the

field (20), as is now commonly done for water

samples, Extending this approach to milk,

juices, and other fluid foods might be feasible.

If some sort of solid sample extraction could
66

be devised in the field, this approach could be

extended to other foods.

In addition to miniaturization, the further development of SPE extractions will reduce or

eliminate the need to use hazardous solvents.

present use of such solvents creates a health

hazard for the chemist and produces a dilemma

for their disposal.

Another spinoff of SPEs is that once the pesticide is on them, they can be stored more easily. The requirement for refrigerating potentially explosive solvents is removed, making the

storage more safe and economical.

(4) Capillary columns. with the exception of

cost, essentially all objections to capillary column chromatography for analysis of pesticides

in foods have been removed. Fifteen-meter-long

capillary columns of the wide bore variety cost

about $250 compared with about $80 for a

packed column, a small difference considering

the potential savings in analysis time (see table 3-2), the availability of guard columns, and

the reusability of columns following a solvent

wash (7).

More important, capillary columns usually

provide lower limits of detection because the

chromatographic peaks are sharper. Lowering

the limit of detection also means that smaller

food samples (100 grams or less) can be analyzed. Once smaller food samples are used, then

analysis becomes more adaptable to robotics.

For capillary columns to become accepted

by many regulatory agencies, additional examination and standardization of columns will-be

needed so that the relative retention time concept of identifying pesticides can be extended

to them. Relative retention time will change

when compared to packed columns, but selection and detailed specifications of capillary

columns and resources to characterize them

fully should relieve this problem. However,

costs in terms of equipment and time could be

great and would have to be considered in light

of existing monitoring activities.

(5) Increased use of HPLC. Since pesticide

metabolizes usually are more polar than their

parent molecule and since HPLC is more adept

at dealing with polar compounds than GC, it

seems that HPLC has potential for analyzing

both the parent and metabolizes simultaneously.

Recent examples include HPLC methods used

for benomyl (fungicide), glyphosate (herbicide),

and metabolizes of fenvalerate (insecticide). All

of the sulfonyl urea herbicides are analyzed by

HPLC with the photoconductivity detector.

The trend toward more polar pesticides among

those under development also makes HPLC

worth examining in the development of MRMs

and SRMs. Detectors are the constraining factor in applying HPLC to pesticide residue analysis in food. While columns are now available

for almost any conceivable type of pesticide,

there is a lack of effective detectors when compared to those available for gas chromatography. Particularly lacking are the element-specific

detectors for pesticides containing atoms such

as phosphorus, sulfur, nitrogen, and the halogens chlorine and bromine, although the photoconductivity detector works for some sulfurand chlorine-containing pesticides.

(6) Use of immunoassay as a detection technique. Using immunoassay for detecting pesticide residues can have several advantages

over conventional methods: They can analyze

an increased number of samples in a given

period, are simpler to use, require less skilled

personnel and comparatively inexpensive equipment, and can analyze samples for less cost

than conventional methods. However, widespread use is constrained by several factors,

indicating that immunoassay will complement

conventional determinative steps for MRMs but

are unlikely to replace them. They may also offer a means to improve SRMs. (See ch. 4 for

a detailed discussion of immunoassay.)

(7) Increased automation. Continued improvements in analytical methods are possible through

automation. Improvements in automation have

focused primarily on the cleanup and detection stages of pesticide residue analysis. Although automating the sample preparation and

extraction steps would generate the greatest

time savings, these steps are also the most difficult to automate because of the many types

of food samples requiring different preparation

(15).
67

Automated equipment including robots involves high capital costs, and many Federal regulatory laboratories may have difficulty purchasing such equipment. Manual procedures

may still be faster than automated ones, although automation may provide other benefits,

e.g., freeing up analysts’ time or reducing analyst exposure to hazardous solvents. Therefore,

decisions to increase the use of automated

equipment must consider the goals of monitoring programs and the moneys available. Robots

are not a cure-all for those regulatory agencies

now inundated with food samples, However,

robots can measurably improve the overall

operation of the analytical laboratory. (See ch.

5 for a detailed discussion of automation.)

(8) MSD. The mass selective detector (MSD)

may have an increasingly important role as a

GC detector in developing MRMs; it is the only

GC detector able to detect any pesticide that

can be volatilized that has a molecular weight

of no more than 650 atomic mass units (20). This

may become an important factor in detector

selection because it would not be constrained

by the need to have a particular atom, such as

sulfur, in the molecule. Although still considered a confirmatory tool, MSD has potential

as a programmable GC detector that can be set

to provide a relatively large degree of selectivity

for pesticides at the trace level. The degree of

applicability of this detector to samples of unknown pesticide application history—the sample types for which the MRMs are designed—

will depend greatly upon improvements in the

number of ion programs that can be used during a chromatographic run.

At present, only eight sets of ions can be programmed into the instrument during a chromatographic run, making its usefulness limited

for MRM work. As use of this type of detector

grows, its purchase cost ($40,000 to $65,000 per

unit, depending upon accessories), should drop

accordingly. Some laboratories have difficulty

justifying such an expensive detection device,

particularity when it is dedicated to quantitative work; typical element-specific detectors

cost about one-tenth this much. Such an MSD

can be used for full mass-spectral scans, however, giving it the capability of being a quanPhoto credit: Food Safety and Inspection Service Laboratory, Athens, GA

Bench-top gas chromatograph/mass selective detector

combinations are used to confirm a violative residue

level in a food sample.

titative and confirmatory tool. MSD devices

have been reduced in size compared with mass

spectrometers of the 1970s and can be placed

on desk or table tops, requiring little more room

than the gas chromatography itself. Space considerations become important when the high

costs of supplying a cool, safe, dust-free environment for contemporary analytical instrumentation are taken into account.

Developing New Method s

(1) New MRMs. Research needs to focus on

the potential for incorporating more pesticides

into existing and emerging MRMs. Significant

metabolizes of these pesticides—often more difficult to detect than the parent compound–

must also be addressed (see box 6-B). As additional data become available, it may become

apparent that existing MRMs need to be modified or new methods developed.

It maybe more advantageous to develop new

MRMs for small numbers of chemically similar

pesticides; this has been done for the phenoxy

herbicides (PAM I); for the pesticides captan,

folpet, and captafol (25); and for the benzimidazole-related fungicides (25). A new MRM is

being proposed for collaborative study for

analyzing the urea herbicides (11). Restricting

new MRMs to such small groups of pesticides

would probably not be as efficient for monitoring purposes as adding to existing MRMs

or developing new comprehensive ones, but it

may provide an interim solution to the ques-
—. — —

68

Box 6-B.—Ongoing Challenges to Methods Development: Metabolizes and New Pesticides

Greatly complicating the issue of developing analytical methods for pesticides in foods is the task

of addressing not only the parent compound but also significant metabolizes. Since metabolism or

degradation of a parent compound generally occurs through cleavage, hydrolysis, conjugation with

sugars or other polar compounds, or oxidation, the products so formed are usually more polar than

the parent and thus more difficult to detect using conventional multiresidue methods (MRMs).

New pesticides may also pose problems for analysis. Forecasts for emerging pesticide types indicate molecular structures that are similar to those seen today; therefore, current MRMs seem adaptable to many new chemicals. However, analytical difficulties may result from lower applications rates

(grams or ounces per acre) of some new pesticides (e.g., the sulfonyl urea herbicides and synthetic

pyrethroid insecticides). While the use of this type of pesticide results in low residue levels, its use

will require more sensitive analytical methods for detection. In addition, many new pesticides have

reduced environmental persistence and therefore rapidly metabolize or breakdown into more polar

products. Also, increased use of non-conventional chemical pesticides, such as microbiological and

genetic and behavioral biochemical, will pose difficulties for analysis and require methods development (18).

Analytical chemists are then faced with the current and growing problem of detecting metabolites. In order to provide a method for determining such metabolize residues, a method that is satisfactory for the parent pesticide may have to be altered; such alterations may include chemical derivatization, changes in the nature of the extracting solvent, changes in the chromatographic determinations

such as different columns and detectors, and sometimes even going to a different mode for the determinative step. Many metabolizes cannot be analyzed by MRMs and will require special procedures (18).

One potential solution to the parent compound metabolize dilemma may be to agree on the use

of “indicator compounds”; these maybe parent compounds or toxicologically significant metabolizes

that have been shown by metabolism studies to exist in a predictable manner under certain environmental conditions. Previous studies may also have shown that the relative amounts of the other associated compounds fall within some quantitative boundaries. Knowing the amount of indicator compound present can therefore provide a semiquantitative idea of the amount of other associated

compounds present. The use of indicator compounds may, in many instances, obviate the need for

using multiple analytical methods to provide information on both parent compound and a list of metabolites (18). Another potential solution would be to develop inexpensive methods to rapidly test whether

such residues exist and will need more difficult analysis performed (18).

tion of how to handle pesticides of widely differ- ess. Since the method will be used for enforceent chemical and physical characteristics.

(2) New SRMs. Developing functional SRMs

is a balance between the use of innovative approaches and the use of techniques that are

practical for regulatory chemists. A successful SRM should be capable of analyzing any

of the toxicologically significant metabolites—

as defined in Subdivision O of EPA’s Guidelines (4)–without separate extractions, cleanup

steps, or analytical columns, certainly without

incorporating another type of detector. Since

most metabolizes are more polar than the parent pesticide, this is a challenge for the method

developer and will slow the development procment only when MRMs are not available, it

should use the same glassware, solvents, reagents, and instrumentation as the MRMs. This

is currently not a requirement of EPA’s Subdivision O Guidelines, but it makes the best use

of available resources, obviating the need for

having infrequently used equipment sitting

around the laboratory.

Frequently, little similarity seems to exist between SRMs presented by tolerance petitioners

for individual pesticides with similar structures.

This situation might be improved if petitioners

made efforts to design “mini-MRMs,” that is,

methods that would apply to more than one pes-
69

ticide, This could be accomplished by making

only slight modifications to one SRM.

Cost Considerations of Methods

Development Research

The costs of sample analysis and research reflect a number of factors. There are the housing and associated upkeep costs for a laboratory that must store toxic and possibly explosive

materials and at the same time must maintain

an environment suitable for sensitive equipment. A range of glassware and solvents, which

must be pure, are required. The sophisticated

instruments are a substantial cost, both in the

initial purchase and in upkeep. GCS when

equipped with detectors and autosamples can

cost $32,000 apiece and HPLCS can average

$25,000 to $30,000. Together they also require

high purity gases or solvents. Service contracts

per instrument can cost $2,000 or more a year.

The other major cost is analyst time, which accounts for a large part of the cost of each analysis. The cost to analyze individual samples has

not been calculated by regulatory laboratories.

The closest approximation may be the price

charged by a private laboratory, where a single MRM analysis may cost hundreds of dollars.

Over the last 30 years as the sensitivity of instruments has improved, their purchase and upkeep costs have increased; therefore, improvements in analysis are often accompanied by

increased costs of analysis (20). New instruments to improve methods also require a high

initial capital expenditure although improvements in manufacturing help control cost. Such

expenses may slow Federal regulatory agencies

from investigating the use of new instruments

for improved methods. A further difficulty is

if such instruments are used to improve methods, field laboratories will also need to purchase

them if these methods are to be used for routine regulatory analyses.

Methods research involves costs beyond those

for sample analyses. First are the tradeoff costs

of doing research. Equipment and personnel

spent on research mean less equipment and

analyst time available for sample analyses.

Therefore, requirements for more research

need to address requirements for current sampling programs, given that changes in one area

can adversely affect the other. Second are the

research resources spent unsuccessfully. In the

process of improving a method or developing

a new one, the analyst attempts to improve the

steps involved in the method. Failures in each

step or in the entire process use up resources

but do not produce results apparent to others.

Third are the costs of validating that an improved method is accurate.

Another factor determining the cost of research will be the goals for improving the regulatory programs. Improving MRMs to analyze

more pesticides and commodities may be carried out in conjunction with regulatory analysis work. But if the focus is on high health

hazard pesticides that cannot be analyzed by

existing MRMs, development of new MRMs

or practical SRMs maybe needed. Longer-term

research, high capital costs, and validation costs

may be required for introducing new technologies for either improving methods or developing new ones. The same may be true for introducing technologies, such as automation and

robotics, that can improve the use of methods.

Goals must be set before the level of research

resources can be determined.

Needs for Adoption and Use of

Methods

Vaiidating Methods

Before any analytical method can be used routinely in the laboratory, it must be validated.

Validation is the process whereby one or more

individual chemists test the suitability of a particular method for collecting analytical data

(21). The suitability of a method will depend

in part upon the circumstances of the application. For example, a method that will be widely

used will require more validation than one

whose use is more confined. The effort expended in validating analytical methods serves

to validate the results of sample analyses. Consequently, method validation at several levels

(e.g., intralaboratory, interlaboratory, and

AOAC collaborative study) is considered inherent to the methods development process.
70

Intralaboratory validation is the lowest validation level. It requires the developer to demonstrate that the method is reproducible, sensitive, specific, and contains all the qualities

needed to meet the method’s analytical purposes. The developer then hands the evaluation of the method to someone else within the

laboratory for further validation.

Interlaboratory validation is the next level.

This level is usually required before a method

is used by other laboratories. The laboratory

developing the method must find another laboratory to test the method and its written description by analyzing samples with unknown

residues and levels. Successful performance of

the method by an analyst other than its developer must be provided before the method

can be sanctioned for use in monitoring.

A more rigorous validation is undertaken for

methods intended for widespread and continuous use. Collaborative study, under the auspices

and rules of the Association of Official Analytical Chemists (AOAC), is a major effort involving six to eight laboratories and is usually

performed for methods that an organization expects to continue using for many years. Methods are usually not studied collaboratively until they have been in use in several laboratories

over an extended period of time and results indicate that they are worth the considerable effort involved. Collaborative studies are far too

expensive to be conducted for all methods. If

the residue measurements produced in a collaborative study meet the statistical requirements for accuracy and precision, the method

is declared official by the AOAC and published

in the Official Methods of Analysis of the

AOAC.

The degree of validation required by FDA and

USDA will differ depending upon the application of the method. For the majority of methods, both agencies require an interlaboratory

validation involving at least two laboratories.

FDA and FSIS encourage the use of AOAC official methods where possible because they are

most widely accepted. All five of FDA’s routinely used MRMs have received AOAC collaborative study for some commodities and

pesticides. In some cases, however, methods

validated by an AOAC collaborative study or

interlaboratory study may not be available, for

example, for pesticides not used in the United

States but found in imports or for applying a

validated method to a new commodity. A lesser

form of validation, such as intralaboratory

study, can be used in such cases. As long as

the analytical results follow well-accepted

principles—sample custody, sample stability,

no false positives in control samples, adequate

recoveries from fortified samples, and confirmation of results—the method can be used for

regulatory enforcement action.

The success of an MRM or group of MRMs

is not necessarily guaranteed by the degree of

formal validation undertaken. For example, the

MRMs used by the California Department of

Food and Agriculture (CDFA) have been developed in-house over the past 20 years as a result

of information from one of the chemical apparatus supply houses (22). No collaborative

studies have ever been done by CDFA (though

one is under consideration now) on their inhouse MRMs, although they have split samples

with FDA laboratories; these split-sample analyses have produced results comparable to those

generated in FDA laboratories (22).

Confirmation of ResultS

When an analysis leads to the finding of a

violation, regulatory agencies require that the

violation be confirmed by a different technique

or method. The most common approach to confirmation is to re-analyze the sample after modifying the original method, for instance, after

changing the detector, column, or sometimes

both (5, 14). In these cases, confirmation does

not require the development of completely new

methods.

A second approach to confirmation has been

identifying a suspected pesticide residue by its

mass spectra. Regulatory agencies are increasingly using mass spectrometers, including the

smaller bench-top types, as detectors for GC,

and in some cases HPLC, for the confirmation

of violations.
71

When violations are found, confirmatory

analyses need to be performed only on certain

samples. That is, if numerous tentative positives are uncovered in a group of samples, confirmation is required on only a representative

part of these samples.

EPA does not require that pesticides receiving new tolerances have confirmatory methods in addition to the method required for monitoring, Consequently, a second battery of

1.

*2,

*3.

4.

5,

6.

7,

CHAPTER 6

Ambrus, A. and Thier, H. P., “Application of

Multiresidue Procedures in Pesticide Residues

Analysis,” Pure and Applied Chemistry, 58(7):

1035-1062, July 1986.

Conacher, H. B.S., “Validation of Analytical

Methods for Pesticide Residues and Confirmation of Results, ” OTA commissioned paper,

Spring 1988.

Ellis, R., “Techniques for and the Role of

Screening Pesticide Residue Analysis, ” OTA

commissioned paper, Spring 1988.

Environmental Protection Agency, Office of

Pesticide and Toxic Substances, “Pesticide

Assessment Guidelines, Subdivision O, Residue

Chemistry,” Washington, DC, October 1982.

Fong, G., Chemical Residue Laboratory, Florida

Department of Agriculture, Tallahassee, FL,

personal communication, May 16, 1988.

Food Safety and Inspection Service, Compound

Evaluation and Analytical Capability National

Residue Program Plan 1987 (Washington, DC:

Food Safety and Inspection Service, January

1987).

Freeman, R, R., and Hayes, M. A,, “Column

Considerations When Doing Trace Analysis on

Open Tubular Columns, ” J. of Chroznatographic Science, 26(4):138-141, April 1988.

8. Hawthorne, S. B., and Miller, D. J., “Extraction

and Recovery of Organic Pollutants from Environmental Solids and Tenax-GC Using Supercritical COz, ” J. of C’hromatographic Science

24(6):258-264, June 1986.

9. Jacobs, W., Enzytech Inc., Lenexa, KS, persona] communication, Aug. 10, 1988.

10. Kovacs, M.F. Jr., and Trichilo, C. L., “Regulatory Perspective of Pesticide Analytical En-

*These reference papers are contained in appendix B.

confirmatory methods does not exist. This suggests that some SRMs might not have confirmatory methods and thus can not be used for

regulatory purposes.

Overall, however, it appears that confirmatory methods will, for the most part, take care

of themselves, assuming that adequate MRMs

will be forthcoming for future pesticides and

that growth in technologies continues,

REFERENCES

forcement Methodology in the United States,”

]. Assoc. off AnaZ. Chem., 70[6):937-940, 1987.

11. ‘Krause, R. T., Center for Food Safety and Ap-

*12.

13.

14.

15.

16.

*17.

*18.

*19.

“20.

21.

plied Nutrition, Food and Drug Administration, Washington, DC, personal communication, May 18, 1988.

Kropscott, B. E., Peck, C. N., and Lento, H. G.,

“The Role of Robotic Automation in the Laboratory,” OTA commissioned paper, Spring

1988.

Lombardo, P., Center for Food Safety and Applied Nutrition, Food and Drug Administration, Washington, DC, personal communication, Aug. 1, 1988.

Luke, M,, Los Angeles Pesticide Laboratory,

Food and Drug Administration, Los Angeles,

CA, personal communication, May 16, 1988.

McCullough, F., ABC Laboratories, Columbia,

MO, personal communication, May 3, 1988,

McMahon, B,M. and Burke, J. A., “Expanding

and Tracking the Capability of Pesticide Multiresidue Methodology Used in the Food and

Drug Administration’s Pesticide Monitoring

Programs,” J. Assoc. Off. Anal. Chenz., 70(6):

1072-1081, NovemberlDecember 1987.

Mumma, R. and Hunter, K,, “Potential of Irnmunoassays in Monitoring Pesticide Residues

in Foods,” OTA commissioned paper, Spring

1988.

Plimmer, J., Hill, K., and Menn, J., “Pesticide

Design: Outlook for the Future, ” OTA commissioned paper, Spring 1988,

Sawyer, L., “The Development of Analytical

Methods for Pesticide Residues,” OTA commissioned paper, Spring 1988,

Seiber, J., “Conventional Pesticide Analytical

Methods: How Can They Be Improved?” OTA

commissioned paper, Spring 1988.

Taylor, S. E., “Pesticide Monitoring Programs:
72

22.

23.

Developing New Methods to Detect Pesticide 24.

Residues in Food,” Report No. 87-413 SPR,

U.S. Congress, Congressional Research Service, Apr. 24, 1987,

Ting, K. C., Anaheim Laboratory, California De- 25,

partment of Food and Agriculture, Anaheim, CA,

personal communication, Apr. 23, 1988.

Trichilo, C., Residue Chemistry Branch, Environmental Protection Agency, Washington,

U.S. Congress, Office of Technology Assessment, “Workshop on Technologies to Detect

Pesticide Residues in Food,” Washington, DC,

Mar. 14-16, 1988.

Walters, S., Pesticide Industrial Chemicals Research Center, Food and Drug Administration,

Detroit, MI, personal communication, May 22,

1988.

DC, personal communication, Apr. 25, 1988. *These reference papers are contained in appendix B.
Chapter 7

Programs for Detecting

Pesticides in Food
Contents

Environmental Protection Agency . . . . . . . . . . .

Methods Submitted for a Pesticide Tolerance

EPA Research.. . . . . . . . . . . . . . . . . . . . . . . . . .

Supporting Programs. . . . . . . . . . . . . . . . . . . . .

Food and Drug Administration. . . . . . . . . . . . . .

United States Department of Agricuhure . . . . .

Chapter preferences . . . . . . . . . . . . . . . . . . . . . .

.

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75

75

76

77

78

81

83
Chapter 7

Federal Methods Development

Programs for Detecting

Pesticides in Food

Successful monitoring of pesticide residues their research most directly affects the methdepends upon the existence of cost-effective ods used for analyzing pesticide residues in

analytical methods for their identification and food. Nevertheless, increased coordination, or

quantification. A number of Federal agencies at least communication, between all Federal

are interested in improving methods to test for agencies involved in such work would help impesticide residues, but only EPA, FDA, and prove use of research resources.

FSIS programs will be discussed here because

Although EPA currently does not have a significant research program on analytical methods development for pesticide residues in food,

EPA supports development of such methods

through three activities:

10

2.

3.

Requiring the submission of a pesticide

residue method as part of the tolerancesetting process for a pesticide to be used

on food or feed and performing a singlelaboratory evaluation of many of the submitted methods.

Performing a small amount of pesticide

methods research that might be applicable to the analysis of food.

Administering several programs that support the ability of Federal and State laboratories to conduct pesticide methods research.

Methods Submitted for a Pesticide

Tolerance

All pesticides for use in the United States

must be first registered with EPA. If a pesticide is to be registered for use on food or feed,

or if its use will result in residues on food or

feed, a tolerance must be first established for

each commodity in which the residue will occur. EPA requires the person or company petitioning for a tolerance to submit an analytical

method able to detect and quantify the pesticide residue in every commodity for which a

tolerance is to be established. The person or

organization petitioning for a tolerance is

known as a petitioner or maybe called a registrant if the registration and tolerance-setting

process occur simultaneously.

EPA’s guidelines allow the submission of either a single or a multiresidue method to fulfill

this requirement (see EPA, “Pesticide Assessment Guidelines, Subdivision O,” October 1982).

The petitioner usually submits a single residue

method (SRM), which is usually less expensive

to develop and more sensitive than a multiresidue method (MRM) (13).

Once a method is submitted, it undergoes a

paper review by EPA’s Office of Pesticides and

Toxic Substances, and then if considered necessary and if resources permit, it is evaluated

by an EPA laboratory. The first one or two analytical methods for a specific pesticide in plant

commodities and one method for that pesticide

in animal commodities normally receive such

an evaluation (24). EPA has two laboratories—

one in Beltsville, MD and one in Bay St. Louis,

MS —that test submitted methods. Methods are

initially reviewed on paper at these laboratories.

75
76

Photo credit: Contractor for the National Aeronautics and Space Administration

EPA has two laboratories that test analytical methods

as part of the tolerance-setting process. Here, an

analyst at the Environmental Chemistry Laboratory

in Bay St. Louis, MS, performs the extraction

step of a method.

If deemed unacceptable, the method is returned

to the petitioner. If the method is acceptable,

an EPA chemist then tests it and makes a recommendation regarding its suitability for enforcing the pesticide tolerance. This process

usually takes 3 months. Where necessary, petitioners will make modifications and resubmit

the method to the Office of Pesticides and Toxic

Substances (16). Once approved, the method

is submitted to FDA for inclusion in FDA’s Pesticide Analytical Manual, Volume II: Methods

for Individual Residues (also known as PAM

II). Copies of the method can also be obtained

from EPA once the tolerance is approved.

The number of methods tested by EPA’s laboratories has been increasing over time and outstripping EPA’s capabilities, resulting in a backlog of methods to be tested and delays in the

registration of some pesticides. For example,

in FY 1986, EPA had 25 methods to test but

was only able to test 19, carrying the rest over

to FY 1987. In FY 1987, EPA was only able to

evaluate 24 of 47 methods that needed testing;

the rest were carried over to the next year. By

March 1988, EPA had 52 methods to test, had

evaluated 17, and expected to receive up to 18

more methods for testing in 1988. In FY 1987,

EPA had assigned 7.5 full-time equivalents (FTEs)

to testing and expected to assign 10 FTEs in

FY 1988, but the backlog still exists (15, 16).

Some SRMs submitted to EPA can be a source

of concern for Federal and State pesticide regulatory agencies, because these methods may

not be practical for use in Federal and State

food monitoring programs for several reasons.

First, these SRMs often involve complex and

time-consuming analytical manipulations. Regulatory agencies prefer to use the more cost-effective MRMs for routine monitoring and to

use SRMs only when intelligence data show

that a pesticide residue maybe present or when

data on the residue level are needed (20). Second, SRMs submitted to EPA need be effective

only for the commodities for which tolerances

are established and may not be applicable to

other commodities (20). Third, some of these

SRMs maybe analytically flawed, poorly documented, or incapable of analyzing significant

metabolizes, making them difficult or unusable for regulatory work (5,17).

EPA has taken several recent steps to improve

the quality and usefulness of submitted SRMs

(25). It is now mandatory for EPA to test all

methods for new pesticides used on foods. User

forms have been included in PAM II to alert

EPA and others of specific problems with a

method. EPA will be requiring an independent evaluation of each method before its submission to EPA beginning August 1, 1989. Also,

EPA now requires petitioners to test whether

their pesticide can be successfully analyzed

through official FDA MRMs, and the test results are sent to FDA. In addition, EPA is considering how to incorporate FDA and USDA

input on the regulatory usefulness of submitted

methods in the tolerance-setting process.

EPA Research

Methods development at EPA is carried out

primarily by EPA’s Office of Research and Development (ORD). Although EPA has identified

pesticide residues in food as a significant source
77

of human exposure to pesticides, ORD does not

have a specific program on methods to detect

these residues because regulation of food is outside EPA’s mandate.

EPA conducts pesticide analysis and some

methods development work at its field laboratories primarily for nonfood matrices. Because

of the different matrices involved, the applicability of EPA research on conventional analytical methods for use on food may be limited,

especially for extraction and cleanup steps. On

the other hand, since EPA uses detection equipment similar to that used in food analysis, EPA

advances in the detection step maybe applicable to food analysis (3, 18).

EPA’s immunoassay work for detecting chemicals may be applicable to food analysis. The

Environmental Monitoring Systems Laboratory

in Las Vegas, NV, houses a 2-year-old program

designed to assess the usefulness of immunoassay for analyzing toxic chemicals, including

pesticides. The program’s budget for FY 1988

is $450,000 and the program’s objectives are

to: develop immunoassays, develop criteria for

evaluating immunoassays, develop a list of

chemicals for which immunoassay can and

should be made, and evaluate commercially

available immunoassay (28). Although none

of this work is aimed at food, immunoassay

developed for analyzing soil and water can be

adapted for use on food. Further EPA research

and the development of an evaluation protocol

for immunoassay could be used by FDA and

USDA. The Las Vegas laboratory has an interagency agreement with USDA to cooperate on

developing antibodies of mutual interest.

Supporting Program s

EPA has several programs that support development of pesticide residue methods at Federal, State, and other laboratories. These programs include the Pesticide and Industrial

Chemicals Repository and a training program

for State laboratories under contract with EPA.

The Pesticide and Industrial Chemicals Repository provides samples of approximately

1,600 pesticides (foreign and domestic) free of

charge to Federal, State, private, and foreign

laboratories. Having a chemical of known concentration and purity, known as a standard, is

necessary to develop new methods and to check

that existing methods used in regulatory work

are correctly identifying pesticides. Standards

are provided by chemical manufacturers, stored

at the repository, and distributed one sample

per chemical per year to a requesting laboratory, although greater quantities will be supplied to Federal laboratories upon request (12).

The repository is funded by three EPA programs: Superfund, Solid Wastes, and Pesticides.

For FY 1988, the repository had a budget of

about $3 million and 22 full-time employees.

The pesticide part of the repository costs about

$630,000 a year to operate. Because the EPA

pesticide program wants to reduce its contribution to the repository’s budget, the repository has recently restricted its distribution of

pesticide standards. As of July 15, 1988, pesticide standards will no longer be provided to

university, private, and foreign laboratories (3,

11).

To further defray costs, EPA is interested in

having recipients such as FDA and USDA, who

are the largest users of pesticide standards, provide additional support for this activity. F DA

is interested in expanding the repository to include an additional 150 foreign pesticides not

registered in the United States but that might

exist on imported foods (12).

In addition to supplying analytical standards,

EPA provides a quality assurance and training

program for State laboratories to improve analysis of pesticides in the environment. Currently,

EPA has contracts with 52 State laboratories

and Puerto Rico, some of which also analyze

for residues in foods as part of their own program. EPA helps maintain the quality of analysis of these laboratories through two means.

First, EPA performs laboratory quality assurance tests by sending out samples containing

known quantities of pesticide residues for the

laboratories to analyze. Second, EPA’s Denver

Regional Office provides weeklong hands-on

training workshops for State personnel on the

use of specific methods. On average, three
78

workshops are held a year with a limit of 12

participants for each. Demand for the workshop

often exceeds available space (10). States have

expressed the need for more such training

courses (2), including training in the area of

analyzing for residues in foods, but this is outside EPA’s training goals.

FOOD AND DRUG ADMINISTRATION

Because of FDA’s mandate to monitor and

enforce pesticide tolerances in food, it is the

lead Federal agency for the development of pesticide analytical methods specifically for food.

FDA’s research concentrates on MRMs as a result of FDA’s need to determine the presence

or absence of many pesticides in food commodities for which little information exists on pesticides application.

In general, FDA’s methods development research can be divided into two broad types: 1)

that which deals with immediate program needs,

and 2) that which is directed to future goals of

greater scope to solve particular problems or

to improve overall effectiveness or efficiency.

Most of FDA’s effort is the first type (7).

Pesticide methods research is primarily conducted in-house at three levels within the agency:

in the 16 field laboratories, in two special research centers, and at the Center for Food

Safety and Applied Nutrition (CFSAN) in Washington, DC. The CFSAN and two research

centers conduct the bulk of methods research

because field laboratories normally spend at

least 90 percent of their time on regulatory

work. FDA does little outside contracting of

methods research, although in late 1987 it

awarded a contract for immunoassay development because of a lack of in-house expertise.

FDA’s pesticide methods research agendas

are planned on a year by year basis. These

agendas are open to interruption as emergencies arise, e.g., the recent EDB problem. 1 Formal planning sessions with headquarters and

field participation are held each year, research

projects are printed in the annual technical

plan, and their progress reported quarterly.

Research priorities are influenced by factors

such as the Surveillance Index, Pestrak, 2 domestic and foreign pesticide use data, new toxicological information, gaps in monitoring coverage, and pesticide registration cancellations.

Mechanisms for setting research priorities are

currently informal, and detailed listings of longrange priorities have not been prepared (14).

No formal list of priority pesticides requiring

research action exists. However, an informal

list of pesticides requiring methods research

in 1988 was developed at the annual planning

meeting for the research centers in 1987. Responsibility for the work was divided among

the research centers and the Los Angeles field

laboratory. The list was a combination of pesticides identified by CFSAN and ongoing work

at the research centers (l). Currently, CFSAN

is developing a long-term research plan to be

completed in 1988. The level of field and outside input into the draft plan is not clear.

CFSAN has the largest concentration of pesticide analytical expertise in one place in the

FDA system, with six FTEs carrying out pesticide methods research for food in its laboratory. CFSAN develops a separate annual technical plan containing its own research projects.

Current research ranges from expanding MRMs

to cover additional pesticides, their metabolizes,

and additional food commodities to evaluating

new technologies and attempting to fit them

into existing methods. CFSAN also develops

new MRMs on pesticides that cannot be tested

with existing MRMs. As the focal point, CFSAN

also provides research direction and advice to

the research centers and field laboratories. Representatives from CFSAN sit on the committees that approve field laboratory research

projects.

llmproved analytical techniques, in 1984, led to the identification of a large number of illegal EDB residues in grain products.

2Pestrak is a computerized data base used to track whether

pesticides can be analyzed using one of FDA’s five routinely used

MRMs.
79

CFSAN has been a leader in the development

of MRMs. Its work has led to the development

of four of the five MRMs routinely used by FDA.

Based on OTA’s observations, however, field

laboratories may not consider the bulk of

CFSAN’s current work to be addressing their

analytical methods needs. In addition, CFSAN’s

research activities do not seem to be meeting

the regulatory needs of field laboratories in a

timely fashion, and because of the lack of a longterm research plan, CFSAN may not be effectively addressing future monitoring needs.

FDA has two research centers developing

methods for analyzing pesticides and other industrial contaminants in foods. Headquarters

and research center staff meet annually to set

the centers’ research agendas, and the centers

request research suggestions from the field laboratories. These two centers—the Total Diet Research Center (TDRC) in Kansas City, MO and

the Pesticide and Industrial Chemicals Research Center (PICRC) in Detroit, MI—were

established in 1980 to meet the analytical needs

of field regulatory laboratories. Combined they

have six FTEs doing research and one FTE for

management.

The TDRC’s work primarily supports the Total Diet Study (TDS) carried out by the Total

Diet laboratory in Kansas City, MO. The Total

Diet Study is not under the same time constraints as many of the field laboratories and

requires more sensitive methods than those

used by the field laboratories. Thus, some of

TDRC’s methods development work done for

the Total Diet laboratory may not be appropriate for field laboratories’ needs.

In several cases, TDRC’s work has benefitted

specific field laboratories; for example, the use

of gel permeation chromatography and widebore capillary chromatography, and the development and expansion of two newer MRMs

that detect a small number of chlorophenoxy

acids or phenylurea herbicides (19). TDRC has

also conducted research assigned by CFSAN

that addresses field laboratories’ specific regulatory needs. For example, TDRC tested a

method for ETU (a breakdown product of EBDC

fungicides) and found it applicable to only a

few commodities (19). TDRC also has a project

that uses mass spectrometry to identify or characterize pesticide residues that cannot be analyzed by conventional chromatographic approaches. This project, however, does not seem

to be well linked with similar efforts at FDA’s

Los Angeles and New York laboratories.

PICRC is the center with primary responsibility for supporting FDA field laboratories’

analytical methods needs for pesticide residue

detection. Much of its work has focused on developing methods for detecting important nonpesticide chemical contaminants in foods, such

as dioxin and PCBs. In the area of pesticides,

PICRC focuses on classes of pesticides not determinable by existing MRMs.

PICRC has developed methods for the detection of captan, folpet, and captafol using widebore capillary gas chromatography and for the

benzimidazole-related fungicides (benomyl,

carbendazin, thiophanate-methyl, and thiabendazole) using high performance liquid chromatography. The latter work was in support

of an assigned field laboratory monitoring program in 1987 and a compliance program in

1988. In response to a contamination problem

in dairy products caused by heptachlor expoxidecontaminated feed, PICRC developed a method

that allowed the Minneapolis District in 1986

to analyze fatty samples more rapidly than by

using the existing official method (27). PICRC

has also worked on applying capillary column

technology to the analysis of pesticide residues

in food and has been instrumental in the Detroit

field laboratory’s adoption of capillary columns

(21). Ongoing research addresses methods for

the detection of the “quats” (paraquat, diquat,

and difenzoquat) and triphenyltin and its metabolites.

As noted, PICRC’s work has supported specific method needs of individual field laboratories. However, OTA observed that several

field laboratories do not find the majority of

PICRC’s work as relevant to their regulatory

needs. In part, PICRC was viewed as not having strong enough ties to the field laboratories,

especially when compared with its ties to

CFSAN. To some extent, the field laboratories
80

contribute to this problem by not working more

closely with PICRC on the type of research

PICRC could conduct. For example, only four

of sixteen field laboratories responded to

PICRC’s request for research proposals for FY

1988 (27).

A total of about seven FTEs carry out pesticide methods research at the field laboratories

through short-, medium-, and long-term work

(6). In the past, medium- and long-term research

were approved by separate committees, but

starting in FY 1989, a single committee composed of laboratory directors, science advisers,

and personnel from CFSAN and the Office of

Regulatory Affairs (ORA) will approve this

work. This committee also will oversee the

work of the two research centers. Much of the

field laboratory research work focuses on evaluating whether additional pesticides can be analyzed through existing MRMs. But field laboratory research can make more significant

contributions. The most widely used MRM, the

Luke method—which was used in 80 percent

of FDA’s 1987 pesticide residue analyses—was

developed at FDA’s Los Angeles laboratory (20).

For short-term research, FDA provides 50

hours of “discretionary research” per operating laboratory analyst. The laboratory director

normally determines how the time will be divided

among analysts and priority topics. “Discretionary research” is used as the need arises for

short-term projects and is often, in the area of

pesticides, aimed at extending an existing MRM

to a new commodity.

With regard to medium-term research at the

field laboratories, a pool of research time made

up of 150 hours per operating analyst is set aside
81

for research projects. Projects usually require

300 hours and might address expanding a known

method to several commodities or developing

a new method for a class of pesticides. Project

proposals from field laboratories, the two research centers, and CFSAN are ranked by a research committee, reviewed by the director and

deputy-director of the ORA (which has line authority over the field laboratories), and final approval rests with the Associate Commissioner

for Regulatory Affairs.

Long-term research at the field laboratories

takes place through the Science Adviser Research Associate Program (SARAP). Field laboratories may contract with one to two persons

from the academic community who then work

with laboratory personnel. Field analysts in

conjunction with the science adviser may propose long-term research projects (6 to 12 months),

often to be done outside the laboratory at the

adviser’s academic institution sometimes with

additional training for the analyst. Approval

must be received from the laboratory, the district, the research committee, the ORA, and the

Associate Commissioner for Regulatory Affairs.

Normally five SARAPs exist at one time, and

they may be for any of the methods areas in

which FDA works (e.g., microbiology), not only

for pesticide residues in food.

A significant amount of resources are used

for pesticide methods research being conducted

by different groups at FDA. However, based

on OTA observations, much of the work conducted by CFSAN and the two research centers

is not adequately supporting the needs of regulatory laboratories for fast, practical methods

for analyzing pesticide residues in food,

This situation could be addressed by existing groups taking the research responsibilities

for which they seem best suited. Normally free

from regulatory “fire-fighting” work and away

from the “front lines,” CFSAN could use its

nucleus of expertise to focus primarily on longterm, future-oriented research. To increase the

regulatory relevancy of the research centers’

work, their research could be more responsive

to field needs as identified by the field laboratories rather than by CFSAN. In support of this,

field laboratories could expend increased effort in communicating their needs to the research centers, especially to PICRC. Although

field laboratories are usually too busy analyzing

samples to conduct research, whatever research

does take place is perhaps the most immediately relevant for regulatory action. Perhaps

more time should be allocated to field laboratory analysts to conduct research, but this

would require additional resources to allow

laboratories to keep up with their regulatory

workload.

UNITED STATES DEPARTMENT OF AGRICULTURE

Development of residue analytical methods method. This emphasis is, in part, a response

by USDA comes under the purview of the FSIS, to recommendations by a 1985 National Acadand pesticide residue research is currently con- emy of Sciences report and a 1987 General Acducted by the Chemistry Division and Field counting Office study (9, 23).

Service Laboratories Division in the Science

Program. FSIS’s pesticide methods develop- FSIS monitors food solely with MRMs and

ment program currently emphasizes the expan- does not depend on SRMS because generally

sion of existing MRMs complemented by the it considers SRMs as too time-consuming and

development of faster methods, many of which expensive to be practical in a large-scale monare based on immunoassay techniques. These itoring program. Therefore, FSIS works primarfaster methods will allow the weeding out of ily to incorporate additional pesticides under

a large percentage of samples that do not have its MRMs rather than to improve SRMs. All

violative residues without having to analyze methods used by USDA in its monitoring prothem with the more expensive conventional grams are subjected to in-house validation,
82

which includes at least three analysts at two

laboratories with a minimum of 12 to 18 analyses per analyst for each pesticide-meat tissue

combination (4).

In 1985, FSIS closed down its central laboratory at Beltsville, MD (a counterpart of FDA’s

CFSAN laboratory), which conducted research

on analytical methods for detecting pesticide

residues in food. Methods research has since

been conducted by FSIS field laboratories,

through contracts with private organizations,

through the Agricultural Research Service

(ARS) of USDA, and through interagency agreements. In addition, the Chemistry Division in

Washington, DC, is also seeking commercially

available rapid test kits.

FSIS has three field laboratories, two of which

are currently working on pesticide residue

methods (located at Athens, GA, and Alameda,

CA). Each laboratory has a methods division

unit, and regulatory personnel may also conduct methods work. In-house work on pesticides totals approximately $200,000 per year

and 5.5 FTEs (8), with the majority of the work

conducted at the Athens, GA, laboratory. Examples of current work include the following:

● Setting up a new, conventional analytical

method for four triazines for regulatory

Photo credit: Food Safety and Inspection Service Laboratory, Athens, GA

Much of FSIS’ pesticide methods research takes place

at the field laboratories. Here, a new methodology

using high performance liquid chromatography

is being evaluated.

●

●

●

work and testing a commercial rapid immunoassay kit to detect triazines in order

to weed out the large percentage of samples that are not violative.

Expanding an existing MRM for organophosphates, and evaluating a commercial,

rapid cholinesterase enzyme kit that tests

for organophosphates.

Setting up a quick semiquantitative immunoassay method for five synthetic pyrethrin

insecticides that makes use of solid phase

extraction for cleanup.

Expanding the number of Pesticides that

gel permeation chromatography can be

used for cleanup of meat products.

FSIS also contracts out research with private

organizations and other Federal agencies. Current contracts for pesticide residue methods total approximately $285,000 and include the following:

●

●

●

The Department of Energy’s Lawrence

Livermore Laboratory’s work on an immunoassay for heptachlor-related chlorinated pesticides. This laboratory also developed the pyrethrin immunoassay now being

implemented at the Athens laboratory.

The Colorado School of Mines is investigating supercritical fluid chromatography

for aniline-based pesticides.

The University of Washington is working

on the use of thin layer chromatography

as part of a quantitative assay.

In addition to outside contracts, ARS also

does work for FSIS on pesticide residue methodologies. Currently, ARS in Peoria, IL, is working on the use of supercritical fluid extraction

and chromatography in low-fat meat products,

and ARS in Beltsville, MD, is researching a

quantitative method using gas chromatography

for the detection of synthetic pyrethroids (26).

The Cooperative State Research Service of

USDA provides funds to four land-grant university laboratories that support the registration

of pesticides for use on minor crops. These laboratories may develop SRMs as part of their

application for tolerances for the pesticide residue on minor crops (22).
8 3

CHAPTER 7 REFERENCES

1. Carson, L., Division of Field Science, Food and

Drug Administration, Rockville, MD, personal

communication, July 14, 1988.

*z. Cusick, W., and Wells, J., “Pesticide Analytical Methods Development at the State Level, ”

OTA commissioned paper, Spring 1988.

3. Delarco, M., Office of Research and Development, U.S. Environmental Protection Agency,

Washington, DC, personal communication,

May 5, 1988.

4. Ellis, R., Chemistry Division, Food Safety and

Inspection Service, Washington, DC, personal

communication, Apr. 6, 1988.

5. Environmental Protection Agency, Office of

Pesticides and Toxic Substances, “Notice to

Manufacturers, Formulators, Producers, and

Registrants of Pesticide Products, ” PR Notice

88-5, 1988.

6. Food and Drug Administration, U.S. Department of Health and Human Services response

to OTA questions, May 1988.

7. Food and Drug Administration, Pesticide and

Industrial Co~taminants Branch, “Develop8,

9.

10.

11

12.

13.

14.

ment of Analytical Methods for Pesticide Residues,” internal document provided to OTA by

FDA, April 1987.

Food Safety and Inspection Service (Office of

Science), U.S. Department of Agriculture, response to OTA questions, Apr. 29, 1988.

Food Safety and Inspection Service, U.S. Department of Agriculture, “IWIS Future Agenda:

Response to the NAS Recommendations,” Washington, DC, June 1986.

Gillis, J., NEIC Center, U.S. Environmental

Protection Agency, Denver, CO, personal communication, May 1988.

Kantor, E., Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency, Las Vegas, NV, personal communication, July 21, 1988.

Kantor, E., Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency, Las Vegas, NV, personal communication, Apr. 29, 1988.

Kimm, V., Office of Pesticides and Toxic Substances, U.S. Environmental Protection Agency,

personal communication, July 18, 1988.

Lombardo, P., Center for Food Safety and Applied Nutrition, Food and Drug Administration, Washington, DC, personal communication, June 29, 1988.

*These reference papers are contained in appendix B,

15.

16.

17,

18.

19,

“20!

21,

Marlow, D., Chemicals Operations Branch,

U.S. Environmental Protection Agency, personal communication, Washington, DC, June

16, 1988.

Marlow, D., Chemicals Operations Branch,

U.S. Environmental Protection Agency, personal communication, Washington, DC, Apr.

14, 1988.

McCasland, W., Texas Department of Agriculture, Brenhan, TX, comment at OTA Workshop

“Technologies to Detect Pesticide Residues in

Food,” Mar. 16, 1988.

Mitchum, R., Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency, Las Vegas, NV, personal communication, May 16, 1988.

Salmon, G., Total Diet Laboratory, Food and

Drug Administration, Kansas City, MO, personal communication, June 29, 1988.

Sawyer, L., “The Development of Analytical

Methods for Pesticide Residues, ” OTA commissioned paper, Spring 1988.

Schneider, L., Detroit Laboratory, Food and

Drug Administration, Detroit, MI, personal

communication, Apr. 22, 1988.

22. Seiber, J., Department of Environmental Toxicology, University of California, Davis, CA,

personal communication, June 16, 1988.

*23. Taylor, S., “Developing Pesticide Analytical

Methods for Food: Considerations for Federal

Policy Formulation,” OTA commissioned paper, Spring 1988.

24. Trichilo, C,, Residue Chemistry Branch, U.S.

Environmental Protection Agency, Washington, DC, personal communication, June 27,

1988.

*25. Trichilo, C., and Schmitt, R., “Federal Pesticide Monitoring Programs: Analytical Methods Development, ” OTA commissioned paper,

Spring 1988.

26. U.S. Department of Agriculture, Agricultural

Research Service, “Annual Report of Research

Programs on FSIS Needs,” Washington, DC,

March 1988.

27. Walters, S., Pesticide and Industrial Chemicals

Research Center, Food and Drug Administration, Detroit, MI, personal communication,

June 29, 1988.

28. Van Emon, J., Environmental Monitoring Systems Laboratory, U.S. Environmental Protection Agency, Las Vegas, NV, personal communication, Apr. 14, 1988.
Chapter 8

Summary and Options
CONTENTS

Page

Reasons for Needing Additional Methods Work . . . ● . . . . . . . . . . . . . . . . . . . . 87

Examples of What Can Be done.... . . . . . . . . . . . . . . . . .. ... ... ..~. .~.. . . 89

Multiresidue Methods . . . . 0 . . . . . . . . . . . .. ... ..~, ....~.. . . . . ....0.... 89

Single Residue Methods .. .. . C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

Semiquantitative and Qualitative Tests .****** ..*****. ● *****+* ● ***.** 91

Validation: Important for Technology/Methods Adoption . . . . . . . . . . . . . . . 92

Findings and Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

Finding l: Federal Agencies’ Pesticide Methods Research, Development

and Adoption Could Be Improved . . . . . . . . . . . . . . . . . . . . . . . . ● ..,0.... 93

Finding 2: Research Coordination and Cooperation Could Be Increased.. 98

Finding 3: Pesticide Methods Submitted for Tolerance Setting

Could Be Improved for Regulatory Use . . ● .***.* . . . . . . . . ● **.,*** ● *. 101

Finding 4: The Quantity and Quality of the Analyst Workforce

Need To Be Maintained . . . . . . . . . . . . . . . . . 0 . . . . . . . . . . . . . . . ........103

Chapter preferences . . . . . . . . . . . . . . . . . ................,.............105

60X

Box Page

8-A, Related Issues . .0......,..,.. . ........+..,.. . . . . . . ........+ . . . . 94

Table

Table Page

8-1. Summary of Options to Improve Federal Detection of Pesticide

Residues in Food . . . * . . . . .+.,..... . . . + . . . . . . . . . . . . . ,~t..,....... 93
Chapter 8

Summary and Options

Regulatory laboratories analyze pesticide These methods provide quantitative data on a

residues in food by one of two ways: large number of pesticides. FDA supplements

its MRM testing with SRMs that also provide

. multiresidue methods (MRMs) or quantitative data. A potential third way to ana-

● single residue methods (SRMs) lyze residues in food is by using rapid semiquantitative and qualitative methods. Currently,

Both the Food and Drug Administration (FDA) the majority of such tests developed for pestiand Food Safety and Inspection Service (FSIS) cides are for nonfood matrices, e.g., water, and

rely on MRMs for the bulk of their testing. are not yet applicable to food.

If analytical testing is to remain the basis of

Federal pesticide residue regulatory programs,

then new and improved analytical methods will

be needed to carry out the mandate of preventing illegal residues in food from reaching the

consumer. The need for improved methods

arises from constraints on existing methods

used today by regulatory agencies in the following areas:

●

●

●

●

●

Coverage: the ability to test for all significant pesticides.

Resources: the availability of sufficient resources (e. g., personnel, instrumentation,

and laboratory facilities) necessary to test

for all significant pesticides.

Confirmation: the ability to verify that a

violation exists.

Regulatory action: the ability to analyze

samples in a timely manner so that violative commodities can be stopped before

they reach the marketplace.

Metabolizes, new pesticides, and inert ingredients: the ability to test for pesticide

metabolities and breakdown products, for

new pesticides having different characteristics than those analyzed using existing

methods, and for significant inert ingredients (if determined necessary).

Analytical methods exist today to analyze for

each pesticide residue on the food for which

a tolerance has been established. These methods, almost all SRMs, are contained in the Pesticide Analytical Manual Volume II (PAM II).

SRMs are not suitable for everyday monitoring of the food supply for several reasons: a

large number of pesticides are commercially

available, more than one pesticide is commonly

applied to a particular commodity, a pesticide

residue may occur on a commodity for which

the commodity has no tolerance, and regulatory laboratories work with samples whose pesticide history is unknown (14).

To maximize coverage with given resources,

FDA and FSIS rely on MRMs for the majority

of their analyses. These MRMs can detect only

certain pesticides that may occur in food, including some pesticides of primary concern to

Federal agencies (31). FDA’s and FSIS’s MRMs

can test for no more than half of the currently

known pesticides. In addition, development of

MRMs has not kept pace with the number of

new pesticides approved for use on food and

feed (14). The five MRMs1 used by FDA can

detect 163 out of 316 pesticides with EPA tolerances and a number of pesticides with temporary or pending tolerances, pesticides with

no tolerances, and metabolizes that could be

found in food (8, 22, see table 3-1 in ch. 3). FSIS’s

four MRMs can detect approximately 40 pesticides and metabolizes of the 227 FSIS lists for

consideration (1, 10).

‘In addition to FDA’s five primary MRMs, several additional

MRMs exist that can analyze a small number of similar pesticides (e.g., chlorophenoxy acetic acids or phenylurea herbicides).

87
88

Some pesticides do not require routine monitoring because they pose low risk to human

health. But not all higher health hazard pesticides can be analyzed through MRMs. Pesticides that can be analyzed by MRMs are linked

through similarities in chemical structure and

behavior, not through the degree of health hazard they pose. The General Accounting Office

found that FDA’s MRMs could not detect 33

pesticides with moderate to high health risks

(3 I). FSIS has identified 10 highly ranked pesticides it would like to monitor routinely but

cannot with its MRMs (11). A number of other

highly ranked pesticides exist that cannot be

analyzed using the MRMs but FSIS considers

them less likely to appear in meat (11).

Many pesticides not detected by FDA’s and

FSIS’s MRMs require the use of specific SRMs

for analyses. SRMs can be as time-consuming

and costly to conduct as MRMs, making them

comparatively expensive and inefficient for

routine monitoring. Thus, they are used sparingly, usually to test for a pesticide known or

suspected to be a problem, to confirm the results of an MRM, or to conduct special surveys

that monitor one-time levels of a specific pesticide residue in food. For some pesticides,

practical SRMs do not yet exist.

When a violation is found, it must be analytically confirmed before enforcement action is

taken. Confirmation is done by analyzing the

sample with a different method or with the

same method originally used but technically

modified. A confirmatory method generally exists for an MRM because modifications can be

made by using a different column and/or detector. Confirmation methods do not exist for

some SRMs and so may constrain their use.

Existing analytical methods, when combined

with sampling and reporting requirements, generally do not provide results fast enough to prevent perishable commodities from reaching the

market even after violations are found. Frequently, it takes considerably more than 2 days

from the time a sample is collected to the time

analytical results are available (31). In many

cases, the food is sold during this interval.

Some pesticides may break down to metabolizes or degradation products hazardous to human health. Analyzing for the parent compound and its metabolizes (or only its significant

metabolizes) may be outside the capability of

existing methods or the available resources of

regulatory agencies. For some older pesticides,

the metabolism data is flawed and significant

metabolizes have not been identified (30). In

addition, existing MRMs are not designed to

detect many polar (water soluble), nonvolatile,

and nonpersistent compounds. Many new pesticides are designed to be less persistent in the

environment than older ones; therefore, they

metabolize or degrade more quickly, producing breakdown products that are more polar

and thus more difficult to detect using existing methods. New classes of compounds, such

as synthetic pyrethroid insecticides and sulfonylurea herbicides, also are not easily analyzed by existing methods, although they should

not be too difficult to detect using available technology (20). This trend suggests that current

MRMs will need to be modified or that new

MRMs be developed to analyze new pesticides

coming on the market.

In addition, pesticides contain a number of

chemicals used for purposes other than pest

control, e.g., colorants and drift control agents.

Currently, these chemicals are categorized under Federal regulation (CFR 180.1001) as “inert ingredients” and are exempted from the tolerance process and Federal monitoring. In

some cases, inerts are potential or known toxic

substances, and increasing attention is being

paid to them as possible health hazards. If Federal monitoring for inerts—or for only those

considered a health risk—is determined necessary, existing MRMs will have to be modified to address the larger number of chemicals

requiring monitoring.
89

EXAMPLES OF WHAT CAN BE DONE

Multiresidue Methods

MRMs will remain the foundation of regulatory analysis. They are superior in terms of cost,

coverage, and quantified data they provide. Several ways exist to improve the use of MRMs:

● Expand the number of pesticides and commodities that existing MRMs can analyze.

● Develop new MRMs for pesticides not detected by existing MRMs.

● Use new technologies to reduce the resources necessary to perform an MRM.

Existing MRMs can be expanded to analyze

additional pesticides as well as additional commodities. The research has not yet been done

to determine whether an existing MRM can be

used to detect a large number of pesticides without modification. Both FDA and USDA are currently conducting research in this area. For

example, FDA’s Los Angeles laboratory is determining if the Luke method can be used to

analyze an additional 80 older, domestic pesticides and 50 foreign ones (15). And FSIS is trying to adapt its MRMs to analyze an additional

seven pesticides (l). The expansion of an MRM

to analyze other pesticides and foods may also

require some methodology modification. In

some cases, subtle modifications such as substituting one solvent for another can increase the

number of pesticides an MRM can determine.

Adding new technologies into an existing

MRM can also expand the number of pesticides

that can be analyzed. For example, new detectors, such as photoconductivity for high performance liquid chromatography (HPLC), can

detect additional pesticides, and capillary

columns for gas chromatography (GC) can separate individual pesticides that normally might

be seen as one peak on a chromatogram from

a packed column. Theoretically, immunoassay

could be used on an extract prepared for an

MRM to identify pesticides the MRM cannot

detect. Thus, immunoassay would function as

an additional detector for the MRM.

Current MRMs might also be expanded by

using different combinations of existing technologies. For example, the ability of the Luke

method to detect a large number of pesticides

is based in part on the use of two to six combinations of packed columns and detectors (15).

Smaller laboratories, however, may not have

the equipment for such combinations. Further

work on other combinations of technologies

could lead to increased coverage of pesticidematrix combinations.

Another way to expand an MRM might be

to develop ways to analyze those parts of the

sample now discarded, e.g., the water and cellulose fractions. Theoretically, some highly polar compounds might end up in the water and

thus would not be analyzed. FDA’s Los Angeles

laboratory found daminozide in the water released during extraction of fruits and vegetables (15). Herbicides can be bound to plant material, not extracted with existing methods, and

lost when cellulose is discarded. The importance of these bound pesticides to human health

has not been established, although initial work

shows that the majority are of little concern (34).

The relative merits of spending resources for

this type of research have not been determined,

but developing techniques for the extraction

of bound herbicides is seen as an expensive research project (34).

Developing new MRMs can help address the

problem of pesticides that cannot be analyzed

by existing MRMs. New MRMs are applicable

to fewer residues than existing MRMs because

such residues will be from pesticides with

widely varying chemical structures. In short,

new MRMs will analyze smaller groups of

chemically related pesticides (25). For example, FDA’s Pesticide and Industrial Chemicals

Research Center (PICRC) is researching how

to expand the use of high performance liquid

chromatography (HPLC) to develop new methods that can identify small groups of pesticides

not easily detected through GC (32).
New methods may also result from emerging technologies. For example, supercritical

fluid extraction (SFE) may simplify sample extraction and cleanup, and supercritical fluid

chromatography (SFC) may improve chromatographic separation. The coupling of SFE/SFC

could lead to new MRMs capable of detecting

small groups of pesticides that are thermally

labile or polar (13). Although not now in regulatory use, SFE/SFC is being researched under

FSIS contracts for use on pesticides and drugs

in meat products.

New technologies may also reduce the time

necessary to perform an MRM, thereby freeing up time for analysis of additional samples,

a broader analysis of each sample, or additional

research. Automated gel permeation chromatography (GPC), autoinjectors, and data processors are already routinely used to free analysts

from time-consuming and tedious laboratory

activities. Solid phase extraction (SPE), automated evaporators, and other technologies are

being developed and used for their ability to

save time during sample cleanup.

Robotics is an emerging automation technology that might free large segments of analysts’

time now spent on repetitive laboratory procedures; robots are expensive, require substantial design modification, and work best for large

numbers of similar samples undergoing the

same analysis. Health and Welfare Canada is

evaluating the use of a robot in a milk survey

for 32 pesticides, metabolizes, and PCBs to carry

out the extraction and cleanup steps. Early results show that the robot can contribute to a

doubled weekly sample output, in part due to

its ability to work at night (17). Some U.S. private companies are also using robots for pesticide residue analysis.

In addition to new technologies, new approaches may also reduce analysis time. For

example, ongoing FDA research on reducing

the size of the sample prepared for analysis

could cut extraction and cleanup times.

Single Residue Methods

SRMs will be required to test for pesticides

that cannot be analyzed by MRMs, especially

those pesticides with significant health hazards.

SRMs can be made more practical for regulatory analysis through improvements in their accuracy, cost, and timeliness.

A first step could be to determine if existing

SRMs (listed in PAM II) are practical and effective. Those SRMs found wanting would be

candidates for improvement or replacement

through Federal research or potentially by the

petitioner who submitted the method. To ensure that new PAM 11 methods did not suffer

from the same problems, EPA could tighten its

requirements for acceptable methods and increase the testing of such methods (for more

details, see Finding 3).

Many of the same technologies available for

improving MRMs or developing new MRMs

could also be used to improve or develop new

SRMs. Technical advances have taken place

since many SRMs were developed. In some

cases, technologies may be more applicable to

SRMs than MRMs. For example, SPEs reduce

cleanup time but may also cause the loss of

pesticides; newer SPEs suffer less from this

problem than older ones. Reducing the loss of

pesticides can improve the usefulness of a technology for an MRM but not for an SRM, since

SRMs only detect one pesticide at a time. Automation and robotics can also be used in SRMs,

especially if an SRM is used to analyze large

numbers of similar samples for a particular pesticide, as in the case of a special survey. Because of insufficient research resources, the

need for improving an individual SRM could

be evaluated on the basis of the pesticide’s

health hazard and the possibility that an MRM

could be adapted to test for the pesticide.

The quantitative immunoassay is an emerging technique that could lead to important new

SRMs, particularly for those pesticides that do

not need extensive cleanup and that cannot be

detected easily by existing analytical techniques
91

or when large numbers of samples need analysis. Quantitative immunoassays’ potential to

analyze more samples in the same period of

time than conventional methods, to lower training and equipment costs, and to potentially

lower analysis costs combine to make their development attractive for analyzing those health

hazardous pesticides that cannot be analyzed

by MRMs. Currently, Health and Welfare Canada is taking steps to implement the regulatory

use of an immunoassay SRM on food that will

give quantifiable results in its field laboratories.

Semiquantitative and Qualitative

Tests

Development of semiquantitative and qualitative tests promises to complement existing approaches while also changing them. Qualitative rapid tests identify a residue if it occurs

at concentrations above a pre-established level,

while semiquantitative methods identify residues over a pre-established concentration and

determine the range of their concentrations.

Both may test for a single pesticide residue or

a group of related ones. In the latter case, the

test can identify only the pesticide group, not

the specific pesticides. The advantages of such

methods are that they can provide fast results

at lower cost than conventional methods and

may be portable. Their disadvantages are that

they usually analyze only a small number of

pesticides, and they do not provide quantitative data.

The lack of quantitative data is a major institutional drawback. Regulatory agencies like

FDA use methods that can detect and quantify

pesticide residues at below-tolerance levels to

collect data on the incidence and levels present

in food. Such quantitative data are used by EPA

in special reviews of pesticides, in pesticide tolerance revocations, and in re-registration of

pesticides and are of interest to other groups.

FDA is concerned that the use of such tests

would adversely affect its data-gathering responsibilities, especially to EPA. To overcome

the quantitative data obstacle, FDA could meet

with EPA to determine EPA’s actual data needs,

how such tests could take place without affecting those needs, and what other data needs

might be filled by such tests (e.g., identifying

that a pesticide residue commonly exists in a

specific food that was thought to be pesticide

free).

A number of possible uses exist for these tests,

which make use of such technologies as thin

layer chromatography, enzyme inhibition, and

immunoassay. In cases of a widespread residue problem in a commodity, these tests might

identify violative samples more quickly and less

expensively.

Secondly, such tests could be used to analyze

large numbers of samples for a particular pesticide or small group of pesticides that are expected to have low violation rates. The small

number of samples identified as violative could

then be analyzed by quantitative methods to

confirm and measure the violation. In this way,

significantly hazardous pesticides could be routinely monitored while minimizing the use of

more expensive SRMs. Along these lines, FSIS

is evaluating a commercial immunoassay kit

for qualitatively analyzing triazines; this kit

could complement a new conventional method

for analyzing triazines that provides quantitative results (l). Where applicable, these tests

might also be applied to sample extracts prepared for an MRM to analyze additional pesticides. FSIS is developing and evaluating several such tests to analyze triazines, synthetic

pyrethroids, and organophosphates in meat

products, and FDA is developing a method

using thin layer chromatography for organotins (1, 32).

Semiquantitative and qualitative tests may

prove applicable for non-laboratory testing if

scientific obstacles can be overcome. The benefits of such an approach would include reducing the costs associated with laboratory analysis (including transporting the sample to the

laboratory) and speeding up identification of

violative samples. Drawbacks would include

the training and equipment needs for the in-
92

specters who would do the testing and the additional time they would need to carry out tests.

FDA has evaluated one commercial kit for onsite use, but the kit produced an unacceptable

number of false negative results (33).

For all these uses of such tests, conventional

methods would have to be used to confirm any

violation detected and to provide assurance that

the tests were not providing unacceptable levels

of false negatives or false positives. Nevertheless, given the pressure for broader monitoring with fixed resources, the appropriate role

of semiquantitative and qualitative rapid tests

in Federal monitoring programs needs to be determined.

Validation: Important for

Technology/Methods Adoption

Validation is an important consideration for

the adoption of any new technology or method

(see ch. 6). Validation is the verification that

a technology or method provides useful analytical data and operates within acceptable performance parameters (for a description of these

parameters see ref. 4). There are several levels

of validation including the following: intralaboratory, interlaboratory, and a collaborative

study. FSIS requires a minimum of three analysts and two collaborating laboratories for validation. FDA requires at least two collaborating laboratories to test the method or technique

but in some cases maybe forced to use an intralaboratory validated method if no other is

available. Validation requires time and trained

personnel—two scarce components of regulatory work.

Collaborative study involves six to eight laboratories under the auspices of the Association

of Official Analytical Chemists (AOAC) and is

the most rigorous form of validation. (Methods

that have been validated in this way are termed

‘‘official” by the AOAC.) The length of time involved to carry out this type of validation (1 to

3 years) (26) and the difficulty in finding enough

laboratories to volunteer their resources restrict

the number of methods validated in this way.

Even the most widely used FDA MRMs are official for only some of the commodities. FDA,

in particular, emphasizes the use of official

methods where possible. The emphasis on official methods and the limits on performing collaborative studies may make it difficult to adopt

a new technology or method if an official one

already exists.

To avoid delays in adopting new technologies

and ensure the availability y of resources for validations, Federal agencies and non-Federal organizations, such as the AOAC, could jointly determine how the speed of official validation

could be increased and how to expand participation of additional laboratories. For example,

private or academic laboratories could take a

greater part in interlaboratory studies.

Immunoassay (discussed in ch. 4) pose a

specific validation need. They will require rigorous validation as a new technology for analyzing pesticide residues, and as they are unfamiliar to most analytical chemists. At the same

time, pesticide chemists’ unfamiliarity with the

technology may make it difficult to find the necessary number of analysts needed to perform

collaborative studies. In addition, collaborative

studies designed for conventional methods may

not be applicable to certain immunoassay applications. Therefore, Federal agencies and

organizations such as AOAC could determine

the protocol for immunoassay validation for

pesticide analysis and promote means to overcome obstacles to their validation.

FINDINGS AND OPTIONS

OTA has identified specific options for im- ● improving Federal agencies’ pesticide methproving the capability of Federal programs to ods research, development, and adoption;

analyze pesticide residues in foods. The options • increasing research coordination and coare summarized in table 8-1 and organized un- operation;

der four categories: ● improving the regulatory usefulness of ana-
93

Table 8-1 .—Summary of Options to Improve Federal Detection of

Pesticide Residues in Food

Improve Federal agencies’

pesticide methods research,

development, and adoption

Increase research coordination and cooperation

Improve the regulatory usefulness of analytical methods

submitted to EPA as part of

the tolerance-setting process

Maintain the quality and quantity of the analyst workforce

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

●

FDAa and FSISb could establish long-term research plans

including priority lists of pesticides requiring improved

methods.

FDA could improve the organization of its research.

GAO could conduct an evaluation of Federal analytical

methods research programs for analyzing pesticides in

food.

Federal agencies could create a methods research and development advisory committee for pesticide residues in

food. The committee could include appropriate non-Federal

representatives.

FDA, FSIS, and EPA could establish a methods workgroup

for pesticide residues in food.

Federal laboratories could increase coordination with State

pesticide residue laboratories.

Federal agencies could improve their use of private sector expertise.

Federal agencies could increase coordination with appropriate agencies of foreign governments.

EPA C could require an independent test of pesticide analytical methods before their submission to EPA.

FDA and FSIS could validate submitted methods.

EPA could require the testing, development, or adaption

of a multiresidue method for any pesticide requiring a

tolerance.

EPA could revise its regulations and guidelines for submitted methods.

FDA and FSIS could review and revise existing methods

cataloged in PAM Il.d

Federal agencies could revise their hiring practices and

find ways to give laboratories increased flexibility in hiring new recruits.

FDA and FSIS could increase continuing education and

training programs for Federal analysts.

FDA and FSIS could sponsor analytical methods training

workshops for State analysts. -

aFDA: Food and Drug Administration of the IJ.S, Departrnerlt of Health and Human Services

bFSIS: Food Safety and Inspection service of the U.S. Department Of Agriculture

C EpA: u.S. Environmental Protection Agency

dpAM Ii: pe~f~~ide Analytical Manual, Volume 11, Washington, DC: Food and Drug Administration)

lytical methods submitted to EPA as part

of the tolerance-setting process; and

● maintaining the quantity and quality of the

analyst workforce.

Although the options could require congressional action, most of the options can be implemented by the relevant Federal agencies

without new or amended legislation. However,

a number of these options would require budget increases or realignments in agencies’ priorities. In addition to improving analytical

methods, the effectiveness of monitoring pesticide residues in food could be enhanced by

addressing related issues (box 8-A).

In general, the barrier to expanding the detection of pesticide residues in food seems to

stem less from the scientific arena than from

the policy one. Individual agencies have given

lower priority to such research because of pressing demands to address other matters. In addition, lack of adequate incentives and resources

slows progress in this area, including the detection of moderate to high health hazard pesticides not now detectable by existing MRMs.

FINDING 1: Federal Agencies’ Pesticide Methods Research, Development, and Adoption

Could Be Improved.

Option 1.1: FDA and FSIS could establish longterm research plans including priority lists

of pesticides requiring improved methods.
94

Box 8-A.—Related Issues

In the processor assessing technologies for the detection of pesticide residues in food, certain

other issues arose that influence the effectiveness of Federal agencies’ monitoring of pesticide residues

in food. Although these issues were outside the scope of this OTA study, they warrant brief discussion

because they affect the technical capability and research direction of the Federal pesticide regulatory

programs. They are the following:

● Intelligence data on pesticide use

● Sampling

● Perception of food safety

Intelligence Data. Analytical chemists can focus their analysis better and improve their ability

to detect pesticide residues if they know what pesticides have been used on the crop. For instance,

FDA’s most widely used MRM, the Luke method, comprises six different column/detector combinations to detect a range of pesticides. Without intelligence data, each of the six combinations would

have to be used to check the food sample. With intelligence data, the number of combinations used

can be reduced to focus on those pesticides thought or known to have been applied to the crop. Having such intelligence data thereby can free equipment for analyses of additional samples.

The lack of intelligence data today is greater for imports than for domestic foods. FDA laboratories obtain information on domestic pesticide use, for example, from State agencies, land grant

universities, USDA’s extension service, domestic growers, and pesticide applicators. Information on

foreign pesticide use is more scarce. FDA uses the Battelle World Pesticides Program database, which

provides country-level data on fungicide, herbicide, and insecticide use in 22 countries. Information

may also be obtained from such sources as foreign agencies making pesticide application recommendations and weather reports. Improvements in intelligence data, especially for imports, would increase the analytical effectiveness of regulatory analyses while improving the use of fixed laboratory

resources. Improved intelligence data would require additional funds and raise the question of who—

regulatory agencies or the private sector—should bear the increased costs.

Sampling. Decisions on how much sampling should take place and what type of commodities

should be sampled affect analytical methods development. For example, a decision to increase sampling could lead to an emphasis on making current methods faster, introducing new and more rapid

methods, and using semiquantitative or qualitative methods to screen out nonviolative samples quickly.

A decision to increase sampling might also lead to requirements for private testing to reduce the

burden on Federal regulatory laboratories. Such a step could require Federal quality control and assurance programs for private laboratories performing the analyses. The resulting increase in private

testing could lead to increased private development of analytical technologies and methods suitable

for regulatory testing.

Correspondingly, a decision to maintain the current level of sampling but increase the number

of pesticides analyzed could promote research on expanding the scope of existing MRMs and on

developing new MRMs and more practical SRMs. A decision to sample a wider variety of commodities might require increased work on adapting existing methods to new commodities.

Perception. A difference of opinion exists with regard to the actual importance of pesticide residues

in food in relation to human health. A significant level of consumer concern and congressional interest exist on the issue. However, the regulatory agencies, FDA and FSIS, do not consider pesticide

residues as a high priority issue for food safety. Most regulatory chemists and laboratory directors

OTA spoke with believe the food supply is safe with regard to the level of pesticides residues present,

and other areas of food regulation should have priority for any new funding. The regulatory agencies’

stand on this point has led to their allocation of fewer resources and incentives for the development

of improved methods for the detection of pesticide residues in food.
95

Federal methods research for pesticide residues in food suffers from a lack of long-term

planning. For example, neither FDA nor FSIS

has a long-term pesticide methods research

plan. Instead, each relies on annual research

plans that include multiyear projects directed

toward short-term program needs. A percentage of research is necessary to address shortterm emergency-oriented research. But regulatory work could benefit from long-term planning designed to provide research direction to

overall needs and monitoring goals. More specifically, a long-term plan would identify potential future problem areas (e.g., emerging

pesticides), develop strategies to address the

problem areas, and forecast resources (i.e.,

skills, time, and funds) necessary to carry out

the strategies. Currently, FDA’s Center for Food

Safety and Applied Nutrition (CFSAN) is developing a long-term research plan to be completed in 1988.

An important element of such a research plan

would be the development of a list of top-priority pesticides requiring improved analytical

methods. To generate such a list would require

developing a formal means to rank pesticides

for regulatory action, ranking the pesticides,

and then identifying those pesticides that cannot be easily detected by existing methods and

those pesticide-matrix combinations for which

existing methods are unsatisfactory. By gathering this information, Federal agencies could

then develop long-term methods research plans

to help identify and measure the most important pesticides of health concern. This list

would also assist State and private researchers in setting research priorities. The priority

list would be subject to continual revision as

new pesticides and new uses were introduced,

older pesticides and their uses were ended, and

new pesticide data (e.g., metabolism and toxicological) were developed. FDA and USDA

may need separate lists because of the different food matrices with which they work. A comparison of those lists with an EPA list of pesticides in environmental matrices would reveal

areas of mutual concern and offer an opportunity for coordination of research.

Much of the work needed for development

of such a list has already been done by the three

agencies. FDA and FSIS have taken steps toward

developing such a list, but a list of top-priority

pesticides requiring methods development is

not available to other agencies nor to the private sector.

FDA began ranking pesticides on its Surveillance Index (S1) in 1981 based on such factors

as pesticide toxicity, production and usage, human exposure, and environmental fate (23). Under the SI, pesticides are placed in one of five

categories of health hazard. The SI was developed primarily to help set monitoring priorities but has also been used to highlight methods research needs. Currently, 205 pesticides

of approximately 316 pesticides with tolerances

have been ranked, with the remainder still to

be considered (9). An additional 120 pesticides

that could be in food will not be ranked because

they have low toxicological effects, do not produce residues in food, are no longer manufactured, or are used only in foreign countries on

foods not intended for export to the United

States (23). Ranking the remaining pesticides

with tolerances (and those pesticides that do

not have tolerances but have high potential to

occur as residues in food) is of some concern

because only 10 pesticides per year were ranked

in 1986 and 1987 vs. the proposed 30 to 50 (9,

23). FDA also has set up a database, Pestrak,

to identify those pesticides that cannot be analyzed through its MRMs.

FSIS has listed 227 pesticides and metabolites of concern and has given each a letter ranking that represents the potential for harmful

residues to occur in animals at slaughter. In

1985, FSIS instituted a new ranking system for

pesticides, the Compound Evaluation System

(CES), using a letter-number ranking code to

represent potential toxicity and human exposure. Of the 227 pesticides, 39 have been ranked

under the CES, though none have been ranked

so far in 1988 (24). Ranked pesticides are then

checked for suitable analytical and confirmatory methods. If highly ranked pesticides are

found not to have suitable methods for their
96

identification and quantification, research priorities can be adjusted to address the problem.

Although the CES is being used to help set research priorities, it is currently too small to provide an overall priority list.

EPA’s need for a list of priority pesticides in

foods is less because its regulatory responsibilities concern the environment, not food (although it has responsibility for some game animals). A similar listing for the pesticide matrices

EPA regulates, however, could be useful to support internal EPA coordination of methods work

as well as coordination with other agencies.

Option 1.2 FDA could improve the organization of its research.

Recent studies of deficiencies in pesticide

monitoring programs point to the need for an

aggressive methods research and development

program (28, 31). FDA conducts the majority

of Federal research on pesticide residue methods for food. OTA has observed that problems

in the organization of FDA’s methods research

adversely affect the agency’s research program.

The basis for OTA’s observations was interviews with persons inside and outside the FDA.

within the timeframe and mandate of this work,

OTA was unable to evaluate FDA’s pesticide

methods research program in depth. Because

OTA’s observations raise significant concerns

about the FDA research program, a more

detailed evaluation of the program as well as

those of FSIS and EPA conducted by the General Accounting Office would be useful to gauge

the importance of OTA’s observations and, if

necessary, to identify remedial actions. While

OTA did not make similar observations of

FSIS’s or EPA’s programs, an analysis of FSIS’s

decentralized research approach, EPA’s methods evaluation process, and the level of coordination with FDA would be important to obtain

an overall picture of Federal methods research

on pesticide residues in food.

Pesticide methods research is conducted by

four organizational bodies within FDA: 2

‘Pesticide methods research is conducted in conjunction with

industrial chemicals methods research, e.g., PCBS and dioxin,

Therefore, the total research FTEs given overstate the number

of persons working solely on pesticide methods.

●

●

●

CFSAN laboratory, which has six FTEs for

pesticide methods research.

Two research centers, the Pesticide and Industrial Chemicals Research Center (PICRC)

and the Total Diet Research Center (TDRC),

which together have six FTEs for pesticide

methods research.

The 16 field laboratories, which together

have approximately seven FTEs for-pesticide methods research.

CFSAN has the greatest concentration of research personnel in one place. Research at

CFSAN has led to development of four of the

five MRMs used by FDA. OTA observed that

FDA field laboratories considered CFSAN’s

current research not geared to regulatory needs,

including the timeliness needs, of field laboratories. Little field involvement seems to exist

in setting CFSAN’s research agenda, which primarily addresses current problems rather than

upcoming issues. CFSAN is the proper body

to prioritize overall pesticide methods needs,

and it does so annually but not in a fashion easily accessible by other Federal agencies, State

programs, or the private sector.

FDA’s research centers—PICRC and TDRC–

were established in 1980 to address the research

needs of field laboratories in analyzing pesticides and industrial contaminants. TDRC’s

primary function is to support the Total Diet

Program, and PICRC’s is to support other regulatory field laboratories. These centers have

developed a number of methods in support of

the field laboratories’ needs, and a number of

specific cases exist of individual field laboratories benefiting from this work.

OTA observed that field laboratories consider

a large part of the research centers’ work as

not being applicable to their regulatory needs.

Again, it seems that field laboratories have little involvement in setting the centers’ research

agendas especially compared to CFSAN. This

lack of field laboratory appreciation for the

centers’ work may in part stem from a failure

of the field laboratories to devote the resources

needed to work with the centers on appropriate research agendas.
97

Pesticide methods research at field laboratories is done primarily through short- and

medium-term projects. The strength of field research is its immediate relevance to problems

at hand. Field research may also significantly

improve FDA’s analytical work. For example,

the Luke MRM was developed at the Los Angeles laboratory, and in 1987 was used in approximately 80 percent of all FDA pesticide

analyses. One weakness of field research is that

the first priority of field laboratories is regulatory work, and if the need arises, research time

will be sacrificed to deal with emergencies. As

noted earlier, field laboratories may not be making the effort to help set relevant research

agendas for the research centers. Field research

also may not be well coordinated between laboratories, leading to duplication of effort or lack

of productive interchange.

Although the current structure of FDA research (CFSAN, research centers, and field research) seems to be workable, OTA believes that

modifications in the responsibilities of each research body might lead to increased productivity results. As mentioned earlier, a long-term

research plan for pesticide methods research

would help FDA address the most important

needs of its regulatory program, develop improved coordination inside and outside the

agency, and address the research responsibilities of FDA’s research organizations. Any plan

should be based on significant input from FDA

field laboratories and from outside experts and

should be subject to modification as new needs

arise.

CFSAN’s research capabilities may be required to address the immediate needs of the

regulatory laboratories. If so, then modifications could help make that work more appropriate for meeting field needs. Possible approaches would be to increase field pesticide

staff involvement in setting CFSAN’s research

agenda or to have CFSAN research staff periodically spend time at the field laboratories to

improve their understanding of field needs.

On the other hand, CFSAN, as a central laboratory with no line authority over the field laboratories and removed from daily regulatory

work, could focus on addressing some longerterm and broader-scope research issues. CFSAN

has the time and resources to identify and evaluate new and existing technologies for their application to pesticide residue analysis. Where

in-house expertise is lacking, such work might

be done through contracts. Contracting allows

access to specific expertise but it needs to be

done in ways that ensure the work is geared

to regulatory needs and is transferable into the

FDA system.

CFSAN also has a key role to play in the area

of methods development for new pesticides.

Changes in the types of new pesticides are seen

as making regulatory work more difficult. By

tracking the development of new pesticides and

addressing the analytical needs for them, CFSAN

could help FDA keep up with its regulatory

responsibilities and avoid possible future crises.

If CFSAN were to be less involved in research

for immediate regulatory needs, then the research centers or the field laboratories might

need additional research resources. Ranking

pesticides that require methods development

research and then coordinating that research

are functions that a central laboratory may be

best able to accomplish. CFSAN’s location enables it to tap EPA’s and USDA’s pesticide data

and research agendas easily, a factor necessary

to carry out coordination of priority setting.

The research centers’ primary purpose is to

support the field laboratories, to be involved

in the day-to-day regulatory needs. One way of

helping to ensure that research centers are doing so would be to involve field laboratory staff

more formally, especially the pesticide specialists, in setting the research centers’ agendas.

In addition, research center personnel could

interact more with field laboratory staff through

personnel exchanges, workshops, and increased

visits to field laboratories, The centers could

assume increased responsibility in technology

adoption by adapting and disseminating to the

field new methods development work done at

CFSAN, at the centers, and at individual field

laboratories.
98

Field laboratories seem best suited to conduct

research that meets their individual regulatory

responsibilities, such as extending existing

methods to analyze additional pesticides and

commodities. They are also able to refine new

technologies or methods for their own particular situation and validate new methods or techniques. In one case, field laboratories have

demonstrated the capability of developing new

MRMs. The modifications detailed above would

demand increased field participation in setting

research agendas for the research centers and

possibly for CFSAN and could require additional research to be carried out by field laboratories. Such a redirection of resources would

require a corresponding increase in pesticide

laboratory personnel to maintain the current

level of regulatory work. Therefore, the expansion of field research would require an overall

increase in FDA’s pesticide methods research

expenditures or an internal redistribution of

research resources.

FINDING 2: Research Coordination and Cooperation Could Be Increased.

The amount of resources available for methods research for pesticide residues in food increases the importance of coordination between

research organizations. Failure to share research plans and results makes it difficult for

agencies to be familiar with each others’ ongoing work and to benefit from that work. Knowledge of what other agencies are doing can help

minimize gaps in pesticide monitoring coverage, facilitate information transfer, provide

early warnings of upcoming concerns, and reduce duplication of efforts. For example, FDA

let a contract to develop monoclinal antibodies for immunoassay without consulting experts at EPA and Health and Welfare Canada.

Antibodies, albeit polyclonal, had already been

developed for two of the pesticides included

in the contract by Health and Welfare Canada

and the University of California at Davis.

Coordination could be increased at all levels:

among Federal agencies; among Federal, State,

and the private sectors; and between the United

States and other countries, in particular, Canada. Resources would have to be redirected in

support of measures to increase coordination.

Means of increasing coordination include the

following:

Option 2.1: Establish a methods research and

development advisory committee for pesticide residues in food.

This committee could include representatives

of Federal and State regulatory programs as

well as representatives of private companies,

agricultural producers, consumers, environmental organizations, academic institutions,

and pesticide registrants. This committee could

include two subgroups: one to deal with policy issues, which would include the chemistry

program administrators, and one to deal with

scientific issues, which would include the principal chemists. This committee could be mandated to advise the Federal government on current problem areas, support the exchange of

results of ongoing government-sponsored projects, and recommend areas of methods research. Another approach that could stand

alone or complement the advisory committee

would be to follow the Canadian approach of

holding workshops with participants from the

relevant groups to address specific methods

needs when a problem emerges (3).

Option 2.2: FDA, FSIS, and EPA could formally

establish a methods workgroup on pesticide

residues in food.

Currently, research coordination takes place

on a formal and informal bases between EPA,

FDA, and FSIS. For example, FDA and USDA

use information provided by EPA to help make

decisions on what pesticides should be monitored.

Currently, a pesticide analytical methods

advisory group with a representative from each

of the three agencies meets on an irregular basis. Past meetings led to improvements in the

methods program, for example, the inclusion

of a user review form in the PAM II to encourage chemists to report problems they had with

specific methods. Overall, the advisory group

does not have authority, resources, nor commitment to coordinate the pesticide residue

methods research of the agencies.
99

Therefore, a more formal workgroup meeting

on a regular basis could improve coordination

of the methods research of the three agencies.

Effective coordination already exists between

FSIS and FDA in another area of regulatory

research—animal drugs. FDA’s Center for Veterinary Medicine and FSIS have a formalized

system for coordinating their work on veterinary drugs through a 2%-year-old working

group that puts research needs in priority, and

coordinates methods research and validation

of new methods (6). In addition, FDA has established project advisory groups with FSIS participation to address veterinary drugs for which

residue methods are not available, to contract

methods research, and to review the resulting

methods (2). Similar coordination on developing research priorities and carrying out methods research and validation could be established between FSIS and FDA in the area of

pesticide residue methods.

As part of the workgroup, EPA could continue to supply data for priority setting and increase its use of FDA and FSIS input to ensure

that methods submitted during tolerance setting are practical for regulatory work. EPA’s

role in coordinating research with the other two

agencies may be smaller but still important.

This is because EPA’s methods research does

not address food and thus may be of less value

to the other agencies. EPA’s extraction and

cleanup processes may not be applicable to

food, especially fatty foods. But since all three

agencies use similar detection equipment, advances in such instruments could be used by

all three agencies (16). Also, immunoassay research on pesticides can be used by the three

agencies because once a pesticide-specific antibody is developed, each agency can then conduct application research to adapt the antibody

to matrices of particular concern. Currently,

FSIS has an interagency agreement with EPA’s

Las Vegas laboratory to promote coordination

on antibodies of mutual interest.

Option 2.3: Coordination between Federal and

State pesticide residue laboratories could be

increased.

State regulatory personnel, like Federal analysts, have firsthand knowledge of the needs

of field laboratories and could provide guidance

for appropriate methods research. Currently,

some Federal-State coordination exists. For example, in some FDA districts, FDA and State

laboratories divide monitoring responsibilities

for certain foods. FDA laboratories also use

State pesticide-use data to decide which pesticides and commodities to test. EPA provides

pesticide standards to State laboratories and

conducts methods training workshops on nonfood matrices for 52 State laboratories. Currently, the California Department of Food and

Agriculture (CDFA) and FDA’s Pacific Coast

Region are developing a Memorandum of Understanding that may include coordination of

methods development and quality assurance

procedures. Further coordination could include

FDA use of State-generated residue data and

training of State personnel in pesticide residue

methods for food by FDA, similar to the program now run by EPA.

Since most State programs are too small to

conduct methods research, opportunities for

Federal-State coordination of research on residue methods may be small. But those States

with small programs are still knowledgeable of

regulatory needs, and their analysts could be

consulted. Several States have large-scale pesticide residue monitoring programs and at least

one, California, has a significant methods research program. The CDFA recently established

a three-person research group to evaluate emerging technologies. It also gave out contracts for

developing three immunoassay for pesticide

residues in the environment. Increased Federal

coordination with State programs like CDFA

would expand the scope of overall methods research efforts. It also would help ensure that

California establish an effective and efficient

program that would complement rather than

duplicate the Federal programs.

Option 2.4: Federal agencies could improve

their use of private sector expertise.

A tremendous amount of research and development on technologies for detecting pesticides in food is conducted by the private sector. The private sector’s contribution to improve

analytical methods for regulatory use could be

increased in the following ways:
100

●

●

Creating incentives to stimulate private research and development of methods.

Tapping the technical expertise of private

industry and academia through training

and technology transfer.

In addition to the private sector’s provision

of analytical methods, primarily SRMs, as part

of the tolerance-setting process, the private sector also develops new and innovative pesticide

residue techniques in response to the market.

Analytical instrument makers innovate partly

in response to Federal analysts’ needs. The development of biologically based technology for

the analysis of chemicals has led to private development and marketing of testing kits.

Federal agencies could promote private sector involvement by making clear their own

needs. Commercial firms would more likely

venture into analytical methods development

for pesticides in food if they perceive that a market exists for their products. By making available a list of what methods are needed (e.g.,

pesticide-matrix combinations) and the type of

method needed (qualitative, semiquantitative,

or quantitative) for regulatory work, Federal

agencies would provide the private sector with

needed research direction and some assurance

that commercial products indeed have a market among regulatory agencies. For example,

FSIS is trying to tap and stimulate the private

development of rapid test kits, although these

efforts may not be focused enough to convince

the private sector that an assured market exists (18). In addition, a common validation procedure would help assure the private sector that

acceptance of the technology would require one

validation study, not several, for new technologies that are to be used by several Federal

agencies.

Along with stimulating market development

of methods, the agencies can provide the private sector with seed money in the form of contracts to encourage increased methods research

and development. With initial seed money for

methods research, the private sector may then

be willing to expend its own money for further

development. Contracts also will allow agencies to take advantage of expertise not found

in-house. FSIS has much of its research done

through contracting, while FDA relies primarily on in-house expertise. With the development

of new technologies, however, agencies like

FDA will need either to increase their contracts

to address new developments or bring the expertise in-house, which may be an expensive

process. The downside of contracting is that

agency personnel must often spend significant

amounts of administrative time developing and

monitoring the contract, It is sometimes difficult to transfer the results directly because of

the nature of regulatory work and lack of inhouse expertise.

A large amount of methods development research occurs at U.S. universities, but much

of it is devoted to the analyses of environmental

matrices, e.g., water, soil, and air, in part because funding is available for such types of

work. These methods are of potential use for

food analysis. Redirection of university research to food might take additional Federal

funding. FDA might improve their use of the

four university laboratories funded by USDA

to develop methods for pesticides requiring tolerances for use on minor crops (27).

In addition to providing seed money for methods development in the private sector and markets for products, Federal agencies can also tap

the expertise that exists in the private sector.

Private sector laboratories of pesticide manufacturers and food processors are ahead of Federal regulatory agencies in the use of certain

technologies, e.g., robotics and capillary columns. They also may have made improvements

in existing methods or developed new techniques of which the Federal agencies are unaware. In some cases, these advances may not

be applicable to regulatory usage. On the other

hand, Federal and State laboratories have

turned to the pesticide manufacturer for advice

on methods after encountering difficulty in pesticide analysis.

Federal agencies could obtain additional methods expertise from the private sector through

joint public/private sponsorship of technical

“hands-on” laboratory workshops. A model

may be the annual pesticide residue workshops
101

sponsored by the State of Florida or the EPA

method workshops held for State analysts and

often taught by private sector personnel using

privately supplied equipment. FDA does tap the

academic community through contracting science advisers from universities at the field laboratories. The majority of science advisers,

however, are not experts on pesticide analytical methods.

Option 2.5: Federal agencies could increase coordination with foreign agencies.

FSIS and FDA have programs whereby foreign scientists work at Federal laboratories to

gain experience in analytical methods and to

pass on their expertise to Federal analysts. Foreign government pesticide regulatory agencies

may have certain methods, research expertise,

and knowledge that might be relevant to the

United States and vice versa. For example,

Health and Welfare Canada has a small pesticide methods research program that seems to

be advanced in several areas such as immunoassay and robotics. Yet little interaction exists

between that agency and U.S. agencies in the

area of immunoassays. Canada has developed

seven immunoassay for use on fruits and

vegetables. U.S. agencies could take advantage

of this expertise and avoid duplicating Canada’s

work. One easy way to facilitate information

transfer between the two countries would be

for U.S. agency personnel to attend the Fall

1988 workshop on immunoassay held for personnel from Canada’s regional laboratories.

FINDING 3: Pesticide Methods Submitted for

Tolerance Setting Could Be Improved for Regulatory Use.

All pesticides used in the United States must

be registered by EPA. If a proposed use of a

pesticide may result in residues in food or feed,

a tolerance (or exemption from tolerance) is required. As part of the tolerance-setting process, EPA requires that the person or organization (known as a petitioner) requesting a

tolerance provide an analytical method that can

be used to enforce the tolerance set for each

pesticide/food combination. To meet this requirement, SRMs, commonly developed to generate data required for pesticide registration

and tolerance setting rather than to meet regulatory needs, are submitted. In some cases,

these methods have not been laboratory tested

by EPA. Thus, some submitted methods have

proved complex, time-consuming, costly, and

sometimes cannot be replicated by another laboratory, thus rendering them impractical for

regulatory work.

Several courses of action exist that could be

taken to ensure that methods submitted for the

tolerance-setting process are more useful for

enforcement purposes. The majority of these

actions, noted below, include stricter EPA requirements on submitted methods to make

them more practical for regulatory work. In

conjunction with any of these actions could be

improved communication between EPA and

the pesticide manufacturer as to what kind of

methods or information would be most useful

for the government. Improved communication

could save the company time by focusing its

chemists’ time, and it could provide agencies

with methods more appropriate for regulatory

work.

Option 3.1: EPA could require an independent

check of pesticide analytical methods before

their submission to EPA.

This requirement would increase the likelihood that a submitted method really works. The

petitioner could contract with an independent

laboratory or have an in-house laboratory carry

out the evaluation (if the in-house laboratory

were not involved in the development of the

particular method). The results from the independent test would be used by EPA in its own

evaluation of the method. This action places

the cost of additional testing on the person petitioning for a tolerance.

EPA has just implemented this requirement

for the first tolerance petition for a pesticide

and for any tolerance petition using a new

method or a significantly changed method. Beginning August 1, 1989, such petitions are required to include the results of an independent evaluation of the submitted method on six

samples (two control samples, two samples fortified with the pesticide at the proposed toler-
102

ance, and two samples fortified at 2 to 5 times

the proposed tolerance). If more than one commodity is being proposed for a tolerance, the

independent evaluation of the method must be

on the commodity most difficult to analyze (7).

Option 3.2: FDA and FSIS could validate methods submitted for tolerance setting.

Increased Federal validation would increase

the likelihood that a submitted method would

be practical for regulatory use. FDA or FSIS

could, in addition to EPA, provide a desk review or laboratory evaluation of all or a selected

number of submitted methods. Criteria for selecting certain pesticides could be based on

such factors as the health hazard of the pesticide, degree of difficulty of the method, or the

use of a new technology or procedure. FDA or

FSIS could evaluate petitioners’ methods as to

their applicability to regulatory work, provide

EPA with results of the evaluation, and if necessary, EPA could require the appropriate

modification of the method before a tolerance

was granted.

This action would place an additional resource burden on FDA and FSIS. FDA probably

would disagree with the redirection of resources

(staff time and funds) for such work, in part

because it depends primarily on MRMs not

SRMs, and thus probably would not consider

validation of petitioner methods a high priority.

Option 3.3: EPA could require the testing, development, or adaptation of a multiresidue

method for any pesticide requiring a tolerance.

FDA and FSIS do not have the needed resources to use SRMs routinely and so depend

upon MRMs for the bulk of their testing. Therefore, it may be reasonable to have petitioners

address the development or adaptation of an

MRM to analyze their pesticide. In 1984, EPA

added the requirement to its tolerance-setting

regulations that petitioners must determine

whether their pesticide can be analyzed by

FDA’s and USDA’s MRMs (40 CFR: part 158.125).

In 1986, FDA made method protocols available

for four of its MRMs, which were needed before such testing could be done and the n e w

regulation became practical (Federal Register,

51(186): 34249, Sept 26, 1986). None of the four

MRMs have to work for the pesticide but the

results must be provided to EPA, which gives

them to FDA. As of May 1988, 12 pesticides

had gone through this procedure. As part of

re-registration, EPA requires registrants to supply similar testing data on older pesticides if

they are not already available. USDA has not

published method protocols for its MRMs, and

therefore methods submitted for tolerances for

meat products have not been tested through

FSIS’s MRMs.

This requirement could be taken one step further by requiring that for all tolerances, or some

subset such as tolerances for new pesticides,

an MRM be developed or adapted for the analysis of the pesticide. While this requirement

would increase the capability of Federal monitoring of pesticide residues in food, it could

also lead to new problems with the regulation

of pesticides and could increase costs for pesticide development.

First, if this requirement applied to all pesticides requiring tolerances, a decision would

have to be made on pesticides now having tolerances that cannot be analyzed through existing MRMs. In some cases, new pesticides that

might have lower health hazards and fewer adverse environmental effects than existing pesticides might not be able to be analyzed through

existing MRMs. The requirement could thereby

slow down or prevent the introduction of safer

pesticides.

Second, the additional research to develop

or adapt an MRM would increase the cost of

pesticide development and might increase the

time before registration is approved. Changes

could be made, however, to prevent the delay

in the pesticide’s entry into the marketplace.

A “conditional registration” could be granted

based on submission of an SRM with the proviso

that the registrant submit an MRM within a

given time period. Because it can take several

years for a new pesticide to achieve widespread

distribution, the MRM would be developed in

time for the routine analysis for that pesticide.

Another time-saving alternative is for EPA to

expedite its review process. Currently, it takes
103

EPA about 12 to 18 months to respond to a submission. Expeditious review, whether through

additional staffing or improvements in efficiency of the review process, could compensate for the additional time required to develop

an MRM.

Option 3.4: EPA could revise its regulations and

guidelines for submitted methods to ensure

that these methods are practical for regulatory work.

EPA has both regulations and guidelines concerning the submission of methods during the

tolerance-setting process. EPA could review

these regulations and guidelines and make

appropriate changes to increase the regulatory

usefulness of submitted methods.

EPA’s regulations require the following: a

method be submitted whenever a tolerance is

required (or for most exemptions to a tolerance),

the method must not be confidential, and the

pesticide be tested through FDA and USDA

MRMs (40 CFR: part 158.125). EPA’s guidelines

(EPA, “Pesticide Assessment Guidelines, Subdivision O,” Oct 1982) include certain technical requirements for submitted methods but do

not carry the same legal weight as the regulations.

The regulations and guidelines could be tightened in a number of ways to make submitted

methods more practical and more appropriate

for enforcement work rather than research. For

example, the regulations could address the need

for practical regulatory methods and either define what is meant by practical or refer to the

guidelines for that explanation. The guidelines

could be rewritten to set stricter limits on the

cost of using a method and the time required

for analysis (possibly to the point of setting maximum times for extraction, cleanup, and detection), and the need to analyze for significant

metabolizes. They could also require improved

and more detailed writeups of the methods, require the use of U.S. measurements, and require

the use of technology easily available to U.S.

regulatory agencies. Currently, revision of regulations and guidelines is not a priority at EPA

and so resources are not available to carry it out.

Option 3.5: FDA and FSIS could review and

revise existing PAM II methods.

PAM II contains the methods submitted during the tolerance-setting process and approved

by EPA. Regulatory analysts have found that

PAM II methods can be impractical to use. The

usefulness of PAM II is further compromised

by a recent EPA decision to discontinue funding (as of FY 1988) for the provision of submitted methods by EPA to FDA for publication

in PAM II.

Several steps could be taken to increase the

utility of PAM II. First is the continuation of

funding at EPA and FDA for PAM II work. Second would be the updating of PAM II by FDA

(which maintains PAM II) and FSIS to winnow

out impractical methods, possibly through desk

reviews and user comments. A further step

would be the replacement of existing methods

with up-to-date methods that are sometimes

available from the pesticide manufacturer. This

work would require FDA and FSIS to redirect

resources. Updating could concentrate on

methods for those pesticides that cannot be analyzed by MRMs and possibly those pesticides

of moderate to high health hazard in order to

be more cost-effective for regulatory needs.

FINDING 4: The Quantity and Quality of the

Analyst Workforce Need To Be Maintained.

Of importance to analytical methods research

efforts is the availability of a high quality workforce. The pool of analytical chemists, however,

is decreasing as fewer students are entering the

field and many experienced chemists (especially at regulatory agencies) are approaching

retirement (26). The concern about the potential shortage of chemists is growing and is

shared by government agencies and the private

sector alike (18).

Option 4.1: Federal agencies could revise their

hiring practices and find ways to give laboratories increased flexibility in hiring new

recruits.

Regulatory agencies are especially hard-pressed

to attract high quality people because of low

starting salaries compared with salaries offered
104

in the private sector. Some smaller State programs are severely affected by this problem.

In addition, Federal hiring guidelines, encumbered by hiring freezes, short windows of hiring opportunity, and long hiring procedures,

seem to make it more difficult to hire people

when they are available (29). The Agricultural

Research Service has partly avoided this problem by having a quick recruitment process for

post-doctoral candidates (19).

To build up this workforce would probably

require active promotion and incentives to

enter the field of analytical chemistry. This

problem is acute in the analytical chemistry specialty of pesticide residue analysis. Many academic institutions that once trained students

in this specialty have left the field due to declines in the availability of research funds

(much of which went to graduate student stipends) or because other areas, such as environmental toxicology or chemistry seemed to offer more opportunities for their students (27).

Some States (e.g., California and Florida) have

already taken steps to attract future chemists

by implementing programs for college students

to work at or visit their regulatory laboratories.

Private industry carries out similar programs.

For example, Proctor & Gamble provides short

courses to undergraduates on careers in analytical chemistry (5). Federal regulatory agencies have similar programs. For example, under an FDA program, undergraduate students

split their year between attending university

and working in a FDA laboratory (12). Federal

agencies could benefit from taking a more active approach to recruit entry-level chemists.

Another way to attract students to the field

would be the establishment of scholarships and

fellowships for undergraduate and graduate

education. To implement this last option would

require additional funding.

Option 4.2: FDA and FSIS could increase continuing education and training programs for

Federal analysts.

Concerns also exist about maintaining the

quality of the regulatory workforce. Many

analysts have been out of school for a number

of years, may not be up-to-date on new developments, and may require some retraining (6).

FSIS now holds an annual workshop on technology development, has a continuing education program and a competitive training program at the University of Georgia, and has

training provided by contractors doing research

(6). FDA has fewer external training connections (one exception is the Science Advisory

Research Associate Program) and provides

most of its training internally. A significant

FDA forum is the annual pesticide workshop,

which brings together pesticide analysts from

the regulatory laboratories, research centers,

and CFSAN. Overall, there seem to be additional opportunities for Federal agencies to

make increased use of private sector and university expertise in staff training. For example,

personnel exchanges with industry and universities could be supported.

Option 4.3: FDA and FSIS could sponsor analytical methods training workshops for State

analysts.

Availability of training is even more important for State laboratory personnel, especially

for individuals in small State laboratories (21).

Analysts in many States do not have the opportunity to learn about the newest advances

in pesticide use and analysis because of lack

of time and funds to attend meetings.

One effective training program for State personnel, administrated by EPA, provides training in analytical methods, though not specifically applicable to pesticides in food. It seems

to be popular and highly regarded by the States,

and there is a waiting list for attendance. A

similar program on pesticide residue methods

could be modeled after EPA’s program and implemented by FDA and FSIS. Currently, only

State personnel near FDA laboratories have the

opportunity to receive FDA training.

Some States also have set up their own training programs. For example, for the last 24 years,

Florida has held a pesticide residue workshop

at which State personnel learn of advances in

the use of methods to detect pesticides in foods

and the environment. Federal and foreign personnel also attend. California is considering
.

establishing a similar workshop for the western States in conjunction with the University

of California.

Support for State analysts training would cost

FDA and FSIS additional resources, but it

105

would improve the overall regulation of pesticide residues in food while supporting closer

Federal-State cooperation.

CHAPTER 8 REFERENCES

1.

2.

3.

*4.

5.

6.

7,

8.

9.

10!

Ashworth, R., Chemistry Division, Food Safety

and Inspection Service, U.S. Department of

Agriculture, Washington, DC, personal communication, May 13, 1988.

Barnes, C., Center for Veterinary Medicine,

Food and Drug Administration, Beltsville, MD,

personal communication, May 16, 1988.

Conacher, H., Health Protection Branch, Health

and Welfare Canada, Ottawa, Canada, comment at OTA workshop “Technologies to Detect Pesticide Residues in Food, ” Mar. 16,

1988 .

Conacher, H., “validation of Analytical Methods for Pesticide Residues and Confirmation

of Results, ” OTA commissioned paper, Spring

1988.

DePalma, D., Proctor & Gamble Company, Cincinnati, OH, comment at the OTA workshop

“Technologies to Detect Pesticide Residues in

Food, ” Mar. 16, 1988.

Ellis, R., Chemistry Division, Food Safety and

Inspection Service, Washington, DC, comment

at OTA workshop “Technologies to Detect Pesticide Residues in F’oods,” Mar. 16, 1988.

Environmental Protection Agency, Office of

Pesticides and Toxic Substances, “Notice to

Manufacturers, Formulators, Producers, and

Registrants of Pesticide Products,” PR Notice

88-5, Washington, DC, 1988.

Food and Drug Administration, Center for

Food Safety and Applied Nutrition, Washington, DC, May 1988 update of Table 1 from

Reed, et al., “The FDA Pesticides Monitoring

Program,” J. Assoc. Off. Anal. Chezn. 70(3):591-

595, 1987.

Food and Drug Administration, response to

OTA questions, April 1988.

Food Safety and Inspection Service, U.S. Department of Agricuhure, Compound Evaluation and Analytical Capability National Residue Program Plan 1987 (Washington, DC: Food

Safety and Inspection Service, January 1987).

*These reference papers are contained in appendix B.

11.

12,

13,

14.

15.

16.

*17.

18.

19

Harr, J., Residue Evaluation and Surveillance

Division, Food Safety and Inspection Service,

U.S. Department of Agriculture, Washington,

DC, personal communication, June 27, 1988.

Hundley, H., Los Angeles Laboratory, Food

and Drug Administration, Los Angeles, CA,

personal communication, July 20, 1988.

Lee, M., Chemistry Laboratory Services, California Department of Food and Agriculture,

Sacramento, CA, personal communication,

June 7, 1988.

Lombardo, P., Center for Food Safety and Applied Nutrition, Food and Drug Administration, Washington, DC, personal communication, June 29, 1988.

Luke, M., Los Angeles Pesticide Laboratory,

Food and Drug Administration, Los Angeles,

CA, personal communication, April 26, 1988.

Mitchum, R., Environmental Monitoring System Laboratory, Environmental Protection

Agency, Las Vegas, NV, personal communication, May 16, 1988.

Newsome, W.H. and Graham, G. F., “Pesticide

Residue Monitoring in Canada,” OTA commissioned paper, Spring 1988.

Office of Technology Assessment, “Technologies to Detect Pesticide Residues in Food, ”

workshop held in Washington, DC, Mar. IA16, 1988.

Plimmer, J., Agricultural Research Service,

U.S. Department of Agriculture, Washington,

DC, comment at OTA workshop “Technologies

to Detect pesticide Residues in Food, ” Mar.

16, 1988.

Plimmer, J., Hill, K., and Menn, J., “Pesticide

Design: Outlook for the Future, ” OTA commissioned paper, Spring 1988.

Polli, R., Vermont Department of Agriculture,

personal communication, Montpelier, VT,

Mar. 7, 1988.

Reed, D., Wessel, J., Burke, J., et al., “The FDA

Pesticides Monitoring Program,” J. Assoc. Off.

Anal. Chem. 70(3):591-595, 1987.

Reed, D., “The FDA Surveillance Index for Pesticides: Establishing Food Monitoring Priori-

*2O,

21,

22.

23.

—
106

ties Based on Potential Health Risk,” ~. Assoc.

off. Anal. Chezn. 68(1):122-124, 1985.

24. Rubin, L., Residue Evaluation and Surveillance

Division, Food Safety and Inspection Service,

Washington, DC, personal communication,

May 16, 1988.

*25, Sawyer, L., “The Development of Analytical

Methods for Pesticide Residues,” OTA commissioned paper, Spring 1988.

26. Sawyer, L., Center for Food Safety and Applied

Nutrition, Food and Drug Administration,

Washington, DC, comment at OTA workshop

“Technologies to Detect pesticide Residues in

Food,” Mar. 16, 1988.

27. Seiber, J., Department of Environmental Toxicology, University of California, Davis, CA,

personal communication, June 20, 1988.

x28. Taylor, S., “Developing Pesticide Analytical

Methods for Food: Considerations for Federal

Policy Formulation,” OTA commissioned paper, Spring 1988.

29, Trichilo, C., Residue Chemistry Branch, Environmental Protection Agency, Washington,

DC, comment at OTA workshop “Technologies to Detect Pesticide Residues in Food, ”

Mar. 16, 1988.

*3o. Trichilo, C. and Schmitt, R., “Federal Pesticide

Monitoring Programs: Analytical Methods Development,” OTA commissioned paper, Spring

1988,

31. U.S. Congress, General Accounting Office,

“Pesticides: Need to Enhance FDA’s Ability

to Protect the Public From Illegal Pesticides,”

GAO/RCED-87-7, October 1986.

32. Walters, S., Pesticide and Industrial Contaminants Research Center, Food and Drug Administration, Detroit, MI, personal communication, Apr. 22, 1988.

33. Wilkes, P., “Evaluation of Enzytec (TM) Pesticide Detection System for Regulatory Use, ”

Laboratory Information Bulletin, Number 8,

August 1987.

34. Worobey, B., Health Protection Branch, Health

and Welfare Canada, Ottawa, Canada, personal

communication, May 5, 1988.

*These reference papers are contained in appendix B.
Appendixes
Appwiix C.

● ● ● * * ● ● ● ● ● ● * ● ● .219

● * * * * * *****..** 230
Appendix A

OTA Workshop Participants

and Observers

OTA Workshop Partlcipants:

Technologies to Detect

Pesticide Residues in Food,

Mar. 14-16, 1988

Henry Conacher

Chief, Food Research Division

Health and Welfare Canada

Ottawa, Canada

John Cowell

Senior Research Group Leader

Environmental Sciences Department

Monsanto Co.

St. Louis, MO

William Cusick

Branch Chief

Chemistry Laboratory Services

Department of Food and Agriculture

Sacramento, CA

Richard Ellis

Director, Chemistry Division

Food Safety and Inspection Service

U.S. Department of Agriculture

Washington, DC

Silvia Canseco Gonzalez

Plant and Animal Health Office

Agriculture Department

Cozoacan, Mexico City, Mexico

Kenneth Hill

Research Chemist

Environmental Chemistry Laboratory

Agricultural Research Service

U.S. Department of Agriculture

Beltsville, MD

Kenneth Hunter

President

Westinghouse Bio-Analytic Systems Corp.

Rockville, MD

Lyle Johnson

Vice President

Laboratory Operations

Analytical Bio-Chemistry

Columbia, MO

Laboratories, Inc.

Bruce Kropscott

Research Chemist

Analytical and Environmental Chemistry

Dow Chemical CO./USA

Midland, MI

Harry Lento

Director, Corporate Analytical Chemistry

Campbell Soup Co.

Camden, NJ

Julius Menn

Associate Deputy Area Director

Plant Science Institute

Agricultural Research Service

U.S. Department of Agriculture

Beltsville, MD

Ralph Mumma

Professor of Chemical Pesticides

Pesticide Research Laboratory

Pennsylvania State University

University Park, PA

W. Harvey Newsome

Head, Pesticide Section

Food Research Division

Health and Welfare Canada

Ottawa, Canada

Cynthia Peck

Senior Research Chemist

Analytical and Environmental Chemistry

Dow Chemical Co./USA

Midland, MI

Jack Plimmer

Research Leader

Environmental Chemistry Laboratory

Agricultural Research Service

U.S. Department of Agriculture

Beltsville, MD

Leon D. Sawyer

Chemist

Center for Food Safety and Applied Nutrition

Food and Drug Administration

U.S. Department of Health and Human Services

Washington, DC

109
110

Jim Seiber

Professor and Associate Dean

Department of Environmental Toxicology

University of California

Davis, CA

Sarah Taylor

Analyst of Life Sciences

Congressional Research Service

The Library of Congress

Washington, DC

Charles Trichilo

Chief, Residue Chemistry Branch

Office of Pesticides and Toxic Substances

Environmental Protection Agency

Washington, DC

OTA Staff

Walter Parham

Susan Shen

H. Anson Moye

Allen Ruby

List of Observers for the Pesticide

Residue Workshop

Raymond Bowers

General Mills

Jerry Burke

Center for Food Safety and Applied Nutrition

Food and Drug Administration

William Cahill

Sandoz Crop Protection

Lou Carson

Division of Field Science

Food and Drug Administration

Jeff Carter

Iowa Specialty Grains

Bernie Chong

Residue Metabolism

Rohn Haas Co.

Marion Clower

Center for Food Safety and Applied Nutrition

Food and Drug Administration

Paul Corneilussen

Center for Food Safety and Applied Nutrition

Food and Drug Administration

Jack Czarnecki

Hoechst Roussel Agri-Vet

Richard A. DePalma

Procter and Gamble

Jim Devine

American Cyanamid

Ed Elkins

co.

National Food Processors Association

Bob Epstein

Food Safety and Inspection Service

U.S. Department of Agriculture

Bruce Ferguson

ImmunoSystems, Inc.

George Fong

Florida Department of Agriculture

Bob Foster

Adolf Coors

Willa Garner

Environmental Protection Agency

John Gillis

EPA/NEIC Center

Denver Federal Center

Environmental Protection Agency

Robert Graves

Environmental Monitoring Systems Laboratory

(Cincinnati)

Environmental Protection Agency

Anthony Gross

Nabisco Brands, Inc.

Janet Hathaway

National Resources Defense Council

Donald Hughes

Hazelton Laboratories

William Jacobs

Enzytech

Ian Kelly

Northern Chemical Co.

Milton Luke

Los Angeles District Laboratory

Food and Drug Administration

Pasquale Lombardo

Center for Food Safety and Applied Nutrition

Food and Drug Administration

William McCasland

Texas Department of Agriculture
111

Albertha Paul

Zymark Corp.

Pete Price

Assembly Office of Research

California State Legislator

Richard Schmitt

Residue Chemistry Branch

Environmental Protection Agency

Susan Shellabarger

Ensys Inc.

Crystal Willis

Grocery Manufacturers of America, Inc.

Albert Yamada

Mike Masaoka Association, Inc.

Congressional Staff

Michael Hoffman

Senator Proxmire’s Office

Debra Jacobson

House Committee on Energy and Commerce

Subcommittee on Oversight and Investigations

Terry Spittler Mary Dunbar

Department of Food Science and Technology Senate Committee on Agriculture, Nutrition,

Cornell University and Forestry

John Scott Warner Jerilyn Hoy

Battelle Cynthia Rasmuseen

General Accounting Office

Richard Wiles

Board on Agriculture

National Academy of Sciences

Robert K. Williams

CIBA-GEIGY Corp.
Appendix B

OTA Workshop Papers

The Development of Analytical Methods

for Pesticide Residues

Leon D. Sawyer, Pesticides and Industrial Chemicals Branch, Division of Contaminants

Chemistry, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Washington, DC

Abstract , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Multiresidue Method Development: Background .

Mills and Mills, Onley and Gaither Methods. , . . .

Storherr Method . . . . . . . . . . . . . . . . . . . . . . . . . . .

Luke Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Krause Method . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Multiresidue Method Development: Summary . . .

Multiresidue Method Development: The Future . .

Impacts of Laws and Regulations . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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The U.S. Food and Drug Administration’s (FDA)

objectives of enforcing tolerances for pesticide

residues in foods and feeds and of determining incidence and levels of pesticide residues in the food

supply are driving forces in FDA development of

residue analytical methods. In turn, such method

development is influenced by development of analytical instrumentation, by changing chemical characteristics of new pesticides, and by the constant

need to analyze additional food samples for more

and varied potential residues.

FDA’s chronology of methods development is

therefore presented as an example of how the availability of appropriate technology can either advance

or hinder development of a needed method, Evolutionary development of five multiresidue methods

is discussed, starting with recognition of an analytical need through effects of available instrumentation or determinative systems, development of extraction and cleanup procedures, verification of

overall method performance, and extension of such

established methods to additional residues and commodities beyond those in the original method study.

Reference to interlaboratory validation of each

method is also included.

Laws and regulations have affected the limits of

determination at which analytical methods must be

valid and have dictated coverage for metabolizes as

well as parent compounds, Future methods development will continue to be driven substantially by

such forces and will include new demands for efficiency in application. The search for improved

efficiency will dictate exploration of such new approaches as immunoassays, rapid cleanup techniques, improved instrumentation, and automation.

Incorporation of these techniques will depend on

the degree to which they prove beneficial in a given

laboratory situation,

The Food and Drug Administration (FDA) samples and analyzes a wide variety of raw agricultural

food and feed products (hereafter referred to as

food) to enforce maximum limits, or tolerances, established for pesticide residues. Commodities sampled do not include meat and poultry, which are

the province of the U.S. Department of Agriculture

(USDA). Residue levels detected, even though they

may be below tolerance levels, are quantified and

recorded in a central data base. Results of analysis

may provide the basis for regulatory actions or may

serve the Environmental Protection Agency (EPA),

World Health Organization and other groups that

112
113

have an interest in the historical incidence and

levels of pesticide residues in food.

Over the past 25 years, the number of samples

analyzed annually by FDA has varied between about

7,000 and 30,000. In fiscal year 1987, about 15,000

samples were analyzed. Also during this year, FDA

responded to 26 separate requests from EPA and

USDA regarding levels and incidence of residues

for 95 chemicals (l). Additionally, FDA’s Center for

Food Safety and Applied Nutrition responded to

28 Freedom of Information Act (FOI) requests for

pesticide monitoring data (1). FDA field offices also

routinely respond to a number of similar FOI requests each year.

Since the early 1960s, the FDA has also monitored

dietary intake of pesticides in a “market basket” of

selected food items (incIuding meat and poultry)

that are purchased at the retail level and then prepared ready-to-eat prior to analysis. Results from

this Total Diet Program provide the only information available in the United States on types and

amounts of pesticides that remain in or on food as

it is consumed. These results are compared with

Acceptable Daily Intakes and serve as a measure

from which food safety judgments can be made. The

data also provide a means to identify trends and

detect isolated contamination sources. In addition

to pesticide residue data, the program also provides

dietary intake information for radionuclides, toxic

elements, essential minerals, and several types of

industrial chemical contaminants. Emphasis, however, has been on pesticide residue data. Bennington and Gunderson (2) and Lombardo (3) provide

in-depth discussions of the history and significance

of this program. Reed et al. (4) discuss the design

and purpose of all FDA pesticide monitoring activities. These references should be consulted for more

details on FDA program goals.

This paper addresses FDA’s analytical methods

development history and does not attempt, in the

brief space allotted, to review the entire field of pesticide residue methods. Analytical methods used in

its regulatory activities include those with the capability to detect and measure several residues in

a single analysis as well as those that detect a single residue or a limited number of related residues.

In most cases, single residue methods are supplied

to EPA by the registrant of a pesticide during the

process of approval for food use. Applicability of

such methods need only be demonstrated on food

items for which a residue tolerance is set. These

methods are published in Volume II of FDA’s Pesticide Analytical Manual (PAM) (5). They often involve complex and time-consuming analytical manipulations. Therefore, resources dictate that single

residue methods are generally used only when it

is known that a pesticide chemical, not recoverable

by a multiresidue method, has been used or when

information on its potential residue level is needed.

In these situations, PAM II methods if applicable

are used in selected surveys. A recent example is

the use of a PAM II method for daminozide in

apples.

On occasion, residue information and regulation

are needed for a pesticide for which no adequate

method exists, e.g., ethylene dibromide (EDB), Resources are then diverted to developing and validating a single residue method. Overall, single residue

methods are employed, out of necessity, to analyze

for selected residues by the FDA and other organizations seeking to test for suspected residues in a

given food or category of foods. Judicious use of

such methods is an important part of the FDA pesticide monitoring program.

Since spray history or environmental background

of most samples is unknown, FDA method development efforts have concentrated on multiresidue

methods. This work has continued for approximately 30 years and has sought to take advantage

of advancements in analytical technologies as they

have become available. Consultation of the scientific literature and contact with other researchers

has allowed FDA to stay abreast of new approaches

to analysis. Continuing interaction among analysts

in FDA’s 16 field laboratories and in headquarters

has led to refinements that have improved reliability and efficiency.

Analytical method studies usually fall into one of

the following categories: development of a new

method or technique; expansion of an existing

method’s applicability to additional analytes and

sample types; integration of new technology into

an existing method; and validation of a method,

technique, or modification. This paper provides an

overview of the historical development of five multiresidue methods and illustrates how FDA’s research in these categories has been applied to evolution of the methodology.

FDA investigators developing residue methods

publish their findings in the scientific literature.

Multiresidue methods most commonly used by

FDA, along with associated supporting information,

are compiled in Volume I of the PAM (5). Once

analytical methods are developed, published, and

proven reliable by a number of laboratories, a more

formal process of validation usually occurs. Collaborative study under the auspices of the Association of Official Analytical Chemists (AOAC) is un-
174

dertaken for selected methods and analyte/food

combinations. Successful collaboration results in

the method’s adoption as “official” and publication

in the AOAC’s Official Methods of Analysis (OMA)

(6).

Hill and Corneliussen (7) have published a comprehensive discussion on the needs and considerations related to official pesticide residue method

validation. They emphasize that validation of methods and changes in methods area long standing regulatory policy. Aside from being a usual laboratory

practice, validation is mandated to ensure that analytical results will withstand scrutiny resulting from

public overview and possible legal actions that may

occur. By regulation (8, 9), both the OMA and PAM

contain official validated methods for regulatory

use. However, the degree of validation for inclusion in the OMA is more stringent than that required by the PAM.

Space in this paper does not permit extensive discussion of the application of methods. Two recent

publications should be consulted, however, for discussions of why FDA develops analytical methods.

Reed et al. (4) describe the goals and strategies of

FDA pesticide programs, and McMahan and Burke

(10) describe the application of analytical methodology in those programs. Topics critical to the valid

application of the methods are covered in the latter; this includes discussions on limits of quantitation and quality assurance in the FDA laboratories.

Muitiresidue Method Development:

Background

Analytical methods for pesticide residues generally require a procedure for extracting residue(s),

“cleanup” procedures to isolate residues of interest from other components, and techniques to measure residue level and confirm its identity. In a review article by Dewey (l), reference is made to use

of pesticide bioassay as early as 1933 (12) for measuring residues of rotenone and its breakdown products. Though this may not be the genesis of pesticide residue analysis, it provided precedence for

extensive research and application of bioassay techniques that continued until about 1960. This means

of determination was both highly sensitive and multiresidue in scope. It also demonstrated good accuracy and sensitivity if a single pesticide residue

were present and its identity known. However, for

samples of unknown spray history, it could only be

used to indicate whether a toxicant(s) was present.

This type of information is of little use for regulatory purposes or for gathering exposure data. Consequently, research activities were initiated to adapt

sample extracts to other sensitive determinative

techniques that would simultaneously offer qualitative and quantitative information. This need was

fulfilled by chromatographic separation followed

by a detection step.

Applied research in chromatographic separation

and detection of multiple pesticide residues provided the greatest impact in evolution of current

methodology. Initial work with paper and thin layer

chromatographic (TLC) systems provided semiqualitative and semiquantitative information. Relatively poor chemical separations were achievable

with a single chromatographic development and

quantitation relied on visual estimations. These procedures were rapidly replaced with gas chromatographic (GC) systems that provided improved qualitative separations and quantitation capabilities with

sensitive (and selective) electronic detectors. TLC

continued to be used but primarily to confirm the

identity of residues first detected by GC.

Ever since GC applications became commonplace,

a continual growth has occurred in detector and

column technologies. Many advances have been incorporated into FDA multiresidue applications only

to be replaced by still newer refinements. It is expected that many of the current GC applications will

again by replaced by capillary column technologies

that currently provide greatly increased separation

capabilities, once the latter are validated or defined

to the extent needed for FDA regulatory purposes.

Multiresidue methods generally include single or

multiple steps to extract, purify (clean up and concentrate) and detect several potential residues simultaneously. During the early developmental stage,

each individual step is tested to illustrate and document its applicability and limitations. This testing is done with all, or at least several, representative chemicals and sample types for which the

method is intended. Continual refinement then follows during routine applications as additional sample types and chemicals are tested. During this time,

changes, additions, or minor modifications in the

originally proposed steps can occur to expand the

method’s applicability.

The continual changes that have occurred in multiresidue methodologies are reflected by numerous

revisions issued for PAM I. This manual was issued

in 1963 and was updated with yearly changes until

1967, In 1968, it was completely revised, and the

second edition was published. Twenty-five major

and minor revisions have been issued since that

time.

In the following discussions, specific examples

of the evolution of the five principal multiresidue
115

methods will be presented. Each method is published in PAM I and the OMA. The OMA version

reflects applicability of the method as it was collaborated. The PAM version offers guidance to additional applications and options. Methods to be discussed are commonly referred to by the name(s) of

the researcher(s) that is (are) recognized as the developer(s) of the originally proposed extraction and

purification steps. The discussions will follow this

convention.

Mills and Mills, Onley and Gaither

methods

These two methods are discussed together because several similarities exist in both their analytical principles and historic development. Collectively, they have been studied more than all other

FDA multiresidue methods combined, and knowledge

gained benefited development of later methods.

The Mills fatty food (13) was originally developed

for determining residues of organochlorine (OC)

pesticides in both fatty and nonfatty food products.

Published in 1959, the method used paper chromatography in the determinative step. The nonfatty

extraction steps were refined and resulted, in part,

in the Mills, Onley and Gaither (MOG) nonfatty food

method (14) in 1963. The extraction and cleanup

steps described for fats, oils, cheese, milk, and animal tissue in the 1959 Mills paper are basically the

same as those currently used for these products.

Both the fatty food and MOG methods use a similar solvent partitioning step and an adsorption

(Florisil) chromatographic purification step to clean

up the extract. The original MOG method provided

two determinative techniques, paper chromatography or GC.

The extensive expansion of these two basic procedures has been reviewed by Burke (15). In his article, Burke chronologically details, with supporting precedent data, the development and evolution

of FDA’s multiresidue methodology from its inception in 1959 to 1970. Most of the paper’s 103 references are related to these two methods and include

21 different studies on variables in the method (e.g.,

Florisil quality, effect of moisture content of sample), 19 method extension reports, and nine AOAC

collaborative studies that eventually led to recognition and expansion in the OMA. There were 24

reports describing GC applications, 11 describing

related identity confirmation tests, and 19 for other

reference purposes,

During this period, the number of chemicals that

were known to be recovered by the original Mills

fatty food method had expanded from 11 to 59 OC

pesticides. Additionally, recoveries of nine organophosphorus (OP) pesticides were documented. The

MOG procedure was initially published with a demonstrated ability to recover 5 representative OC

pesticides from 11 products. By 1970, the recoverability of 84 pesticide (and related) chemicals was

documented; 15 of these were OP pesticides. The

combined methods were known to be applicable to

about 450 different food products.

By 1970, the determinative step for both procedures had evolved from semiquantitative paper

chromatography to quantitative GC determinations

with an ever-expanding selection of element-specific or element-selective detectors. During 1959-

1970, the following detectors were developed and

applied to pesticide analysis: microcoulometric,

electron capture (EC), alkali flame ionization (KC1TD)

and its simultaneous operation with EC, flame photometric (FPD), and electrolytic conductivity. Mass

spectrometry was also applied for confirmation of

residue identity. Also during this period, GC behavioral characteristics of many pesticide chemicals (at specified conditions) were determined and

tabulated to aid in identifying GC responses. This

was accomplished primarily with two general purpose GC columns, but other specialty columns began to be developed for difficult separations and

difficult-to-chromatograph polar chemicals.

Since 1970, expansion of the methods’ proven capabilities has continued with five AOAC collaborative studies (16-20) and recovery information for

additional pesticides and commodities. New chromatographic technologies continue to be introduced

and older ones replaced. The methods have also

been expanded for use in determining residues of

industrial chemicals (e.g., polychlorinated biphenyls).

Currently, PAM I Appendix I lists approximately

160 chemicals that are partially or completely recovered by the Mills fatty food method and approximately 215 by the MOG.

In 1987, of the 15,592 food and feed samples (21)

analyzed by FDA laboratories, approximately 18

percent (2,827) were analyzed by one of these two

methods. Usage and expansion are expected to continue, particularly with feed materials and fatty

foods, These methods, originally designed for nonpolar OC compounds, do not recover many of the

currently used pesticides and their metabolizes. This

limitation led to development of the Storherr method

for the OP class of pesticides.
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Storherr Method

As noted earlier, some of the OP class of pesticides are recovered quantitatively by the MOG

method. However, many are polar or reactive and

consequently are not recovered through the partitioning and/or Florisil cleanup steps of that procedure. Also, because of their polar or reactive nature,

the OP pesticides are more difficult to determine

by GC than the nonpolar OC pesticides.

The Storherr method is applicable to low and high

moisture nonfatty foods (e.g., fruits, vegetables,

grains) and, like the MOG method, it evolved from

previous procedures designed for fly bioassay, paper chromatographic, and TLC determinative steps.

Although method development for OP pesticides

was being conducted concurrent with that for OC

pesticides, researchers lacked selective GC detectors that were available for OC pesticides in the early

1960s. In 1964, Giuffrida (22) introduced the KCITD,

which was both sensitive and selective to OP chemicals. In the same year, Storherr et al. (23) published

a method for OP determinations using this detector. The method demonstrated the detector’s utility, but it did not extend recoverability to any chemicals beyond that achievable by the MOG procedure.

Consequently, the detector was connected with the

EC detectors used for OC analysis so simultaneous

determination of some OP pesticides could be made.

Thus, used in this way, the early Storherr method

was an extension of the MOG method.

As Storherr et al. (23) noted, GC determination

of the more polar OP pesticides was not possible

at that time without development of different types

of GC columns. In two separate studies in 1966 (24)

and 1968 (25), GC columns containing diethylene

glycol succinate (DEGS) were demonstrated to be

compatible with polar OP pesticides. Storherr and

Watts (26) investigated chromatographic properties

of more than 60 OP and metabolize chemicals with

DEGS and the commonly used silicone liquid phase

columns. In a companion paper (27), the DEGS

column was described for determining recoveries

of highly polar OP chemicals in a method that used

an ethyl acetate extractant and a charcoal column

cleanup.

In 1971 (28) Storherr et al. changed the extraction step of the previous method so that it was identical to that used with the MOG. This improved

overall analytical efficiency by enabling analysis for

a wider variety of OP pesticides from a portion of

the same extract prepared for MOG analysis. This

method was collaboratively studied in 1974 (29) and

is published in the OMA. The collaborative study

also demonstrated equivalent performance of KC1TDs

and the newer FPDs that have been introduced for

phosphorus selective detection in 1966 by Brody

and Chancy (30). Unfortunately, determinations

with DEGS columns could not be included in the

collaborative test of the method because this material was not manufactured in a uniform manner;

consequently its chromatographic performance

proved extremely variable.

Prior to development of this method, other developments occurred in OP methodology that are still

of interest. A study of the variation in different charcoals (27) set precedence for the cleanup step used

in the Krause(31) method for N-methyl carbamates

and an ancillary cleanup step in the Luke et al, (32)

method. A distillation method of sample cleanup

(sweep-codistillation), was developed (33) and collaboratively studied (34). The method was also investigated for use with OC pesticides (35, 36) and

is of current interest because of recent commercial

development and claimed efficiency (37). The commercial system, Unitrex@

, is undergoing evaluation

for FDA applications in multiresidue analyses.

The Storherr method had its most extensive use

in FDA’s Total Diet Program after modifications (38)

were made to achieve lower limits of quantitation.

Its application in the Total Diet Program and other

FDA pesticide programs for high moisture products has now been essentially replaced by the Luke

method. The method was referenced for use in only

13 analyses by FDA in 1987. The Luke method has

also essentially replaced use of the MOG procedure

for analysis of fresh fruits and vegetables.

Luke Method

This method, in one variation or another, was

used in approximately 80 percent (11,922) of the

15,592 1987 FDA pesticide residue analyses. The

evolution of this method’s applicability and general

acceptance has been in direct relationship to advances in GC technology since 1975.

The method (32) was proposed by FDA’s Los Angeles pesticide analytical group and was designed

to recover essentially all nonionic pesticides in the

OC, OP, organonitrogen (ON) and hydrocarbon

(HC) classes. The approach uses an acetone extractant, minimal cleanup and various GC systems with

element-selective and element-specific detectors.

The initial method determined residues of the OP

and ON classes in a crude extract obtained after

a solvent transfer step. These classes were to be determined with the KC1TD detector and use of two

GC columns with methyl silicone and DEGS liquid

phases. Separate portions of the extract were cleaned

up with a modified MOG Florisil step prior to OC
117

and HC determinations by GC with EC and flame

ionization detectors, respectively. This approach

could recover 15 OP, 9 OC, 5 ON, and 2 HC pesticides.

The major advantage of the Luke method when

it was first proposed was an increase in efficiency

of sample work up. Most chemicals initially studied could be recovered by existing multiresidue

methods of Storherr, MOG, and Holden (39). The

improvement in efficiency resulted from the modified MOG Florisil cleanup and substitution of acetone for acetonitrile (common to Storherr and MOG

methods) as the extractant. Acetone eliminated the

exhaustive concentration steps necessary for removing traces of acetonitrile if a KC1TD (acetonitrile sensitive) was used.

The Luke method was not immediately adopted

outside the Los Angeles laboratory, however. Since

FPDs were replacing KC1TDS in general use for OP

determinations, residual acetonitrile was of diminishing concern, and efficiency claimed for the

method seemed minimal. There also was an initial

reluctance among chemists to subject GC systems to

the crude sample extracts obtained by the method.

By 1977, several FDA laboratories realized the potential of this approach, and in 1978 the method was

published in PAM I. However, the GC determinative steps were not well defined or rugged. Later

in 1978, the first of several interlaboratory studies

was initiated to standardize GTC conditions for use

with this procedure. The first study addressed the

troublesome DEGS chromatography (discussed in

the Storherr method) with FPD detection. Satisfactory reproducibility was obtained with an improved

quality of commercially available DEGS. Other

studies with fortified samples in 1979, 1980, and

1981 showed that overall interlaboratory performance of the procedure was acceptable.

This method was further refined in 1981 when

Luke et al. (40) reported that a satisfactory substitution of the EC detector could be accomplished with

a newly designed Hall electrolytic conductivity detector for OC pesticide determinations. This refinement eliminated the need for Florisil cleanup and

further increased the efficiency of analysis along

with the potential for expanding recovery to additional compounds. After a successful interlaboratory study (41) of this detector’s performance was

completed, the method was successfully collaborated in 1983 (42) and was published in the OMA.

This AOAC study included six pesticides that represented both OC and OP classes of pesticides.

These are the only broad classes of chemicals for

which the GC determination has AOAC official status, but the method is adaptable to any number of

specialized determinative steps. The extraction and

cleanup steps of this method have recently been

proven adaptable to the multicarbamate detection

of the Krause method (31).

Krause Method

This method is unique among the other multiresidue methods mentioned. It introduced high performance liquid chromatography (HPLC) for separation and fluorescence spectroscopy for detection.

The HPLC method was developed after several GC

approaches were investigated and considered inadequate for analysis for this class of pesticide

chemicals.

FDA began monitoring for residues of one highly

used carbamated insecticide (carbaryl) in the mid1960s. The method was a semiquantitative TLC procedure (43) that also determined one carbaryl metabolite. In 1973, Holden (39) published a multiresidue

method with a GC determinative step that recovered 13 chemicals of the carbamate class. It used

the same extraction step as the MOG procedure,

and GC conditions were basically those used for OC

pesticide determinations; however, it required that

residues be derivatized in order to be detected by

the GC system. The method was officially collaborated in 1974 (44) and published in the OMA. For

the most part, method performance was satisfactory. However, it was lengthy and interferences

were common, A purified derivatization reagent

was needed, and proper GC conditions were difficult to maintain. It also failed to recover some

metabolizes and two of the most widely used pesticides of this class, benomyl and methomyl, These

two pesticides are thermally unstable and not

amenable to GC analysis.

To overcome these inherent problems, Krause (31)

adapted the HPLC approach pioneered by Moye et

al. (45) for the determinative step. Besides HPLC

separation and fluorescence detection, this approach

featured a unique two step, in-line chemical reaction

and derivation process. In developing the total

method, a modification of a partitioning step used

in Holden’s procedure and a charcoal column

cleanup based on the Storherr method were included. The extraction step was extensively studied and validated (46, 47) with 14C labeled carbamate pesticides that were field-incurred. Another

feature of the method is a refrigerated rotary evaporation step, which minimized losses attributable to

thermal degradation.
118

After this method became generally available, it

was 3 years before the method could be collaboratively studied. This time was needed so that a sufficient number of laboratories could obtain needed

equipment and develop necessary expertise. The

collaborative study was completed in 1984 (48) and

the method is in the OMA.

This method is capable of recovering approximately 16 parent and metabolize chemicals of the

N-methyl carbamate class. It also has shown the

ability to recover certain other chemicals (49). In

1987, FDA analyzed only 34 samples by this method

in its entirety, but the HPLC detection step was used

with 588 other samples. Currently, the faster Luke

sample work up is usually used in place of that initially researched and collaborated. A recovery study

that supports the validity of this combination of

methods has been completed (50). The primary use

of the complete Krause method is to confirm levels

of regulatory significance when found by the rapid

approach.

Multiresidue Method Deveiopment:

Summary

These necessarily brief discussions of the most

widely used FDA multiresidue methods exemplify

the constant evolution that has occurred, and is

occurring, as new technologies are made available

and experience with method performance is gained.

They also illustrate the historical time that has

elapsed from the first proposal of a method to completion of a successfully collaborated official method,

about 10 years. By the time the Storherr, Holden,

and Krause methods had gained official status, they

were already being modified or preferentially

replaced by more efficient procedures. The popular Luke method has been modified for use in FDA’s

Total Diet Program (51). [Note: Total Diet multiresidue methodology development and evolution

have roughly followed those of the general methods, but this methodology is specialized enough that

it warrants a separate discussion, which is not included here. The previously referenced (z) review

article of the 26-year history of this program should

be consulted for further details.]

Expanding the number of compounds recovered

by multiresidue methods provides FDA with improved coverage of potential residues within existing monitoring programs. For this reason, FDA has

committed resources every year to testing additional chemicals through existing methods. A computerized system, called Pestrak, has been developed to track the current status of data about

compounds known to be recovered through each

of the methods discussed here (10).

The constant hybridization of methods has made

it difficult to describe which chemicals are recovered through any particular methods. Certain variations in all the basic methods can be, and are, employed to address particularly difficult analyte/food

combinations. This may be accomplished through

variation in any of the steps of the method, such

as changing the extraction, modifying the cleanup,

use of special GC columns or detectors, etc. Validation of the resulting method variation is an integral part of the process. FDA currently defines

analytical method codes for 59 individual extraction/cleanup variations and 23 determinative steps

for recording multiresidue method analysis results

in its residue data system. Up to 20 of the extraction/cleanup codes apply to the MOG procedure

alone. Specific knowledge of the capabilities of each

of these steps and of exactly how they were applied

determines the recovery capability of an analysis,

not of a method per se.

Multiresidue methods are often criticized for their

inability to produce rapid regulatory answers for

samples collected for monitoring purposes. In reality, these methods, with modifications, are readily adaptable to provide this type of information

when a specific pesticide/commodity problem has

been identified or is suspected. In these situations,

it is also not uncommon to utilize less formalized

methods such as those found in FDA’s Laboratory

Information Bulletins or the scientific literature to

facilitate rapid analyses. Much of the analytical data

generated under these circumstances is semiquantitative. Examples of such rapid testing occurred

in two recent widely publicized misuse situations:

aldicarb in California watermelons and heptachlor

metabolizes in milk from an Arkansas dairy shed.

Application of such techniques, as used by FDA

laboratories in the above instances, greatly increased

sample throughput. However, this practice fails to

detect other potential residues present in legal or

illegal amounts. Since illegal residues occur in only

a small percentage of samples, and other residues

are routinely detected, classical multiresidue analytical approaches provide a better measure of the

total pesticide residue burden in the food supply.

Usage of such methods is applicable for those specific pesticide/commodity situations in which there

is an identified need for rapid analysis and such

analyses are carried out on a planned and coordinated basis to allow proper interpretation of the

findings.
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Multiresidue Method Development:

The Future

Method development for pesticide residues is expected to continue evolving as it has in the past;

researchers will apply and adapt technology, as it

becomes available, to meet the needs resulting from

pesticide usage and environmental contamination.

Multiresidue methods are still the most effective

way to examine food samples of unknown treatment

history and so they will be used where applicable.

Existing methods will continue to be used and expanded wherever practical. Special attention will

be given to use of new determinative techniques.

However, new methods for residues not amenable to existing methods must be developed, and

these will be multiresidue methods wherever possible. The method proposal by Clower for determination of a number of volatile fumigants (52) is an

example.

New methods will be applicable to fewer residues

than most of those described here because they involve chemicals whose structures vary widely and

preclude easy separation and detection by today’s

technology. Method development for chemicals not

recovered by existing methods may well follow the

approach taken in developing the Krause method,

in which a very selective determinative step was

developed to focus on a relatively small group of

chemically related residues.

Current examples of this approach include the

Hopper method for chlorophenoxy acetic acid

residues (53), the Luchtefeld method for phenylurea

herbicides (54), and an ongoing effort within FDA’s

Pesticides and Industrial Chemicals Research Center to develop methods for compounds with benzimidazole structures, for the “quat” family (paraquat, etc.), and for organic tin compounds. Within

the Division of Contaminants Chemistry, work continues to develop methods for residues with substitute aniline and nitro aromatic structures.

Technologies currently available and being tested

for adaptability in multiresidue methodology include selective HPLC detection using photoconductivity and electrochemical detectors, capillary column chromatography, and simplified cleanup steps

such as solid phase extraction and distillation

(Unitrex) techniques. Attempts continue to find a

stable and reproducible GC detector that is selective for ON compounds. Other technologies yet to

be applied broadly in residue monitoring include

supercritical fluid chromatography and immunoassay techniques.

Certain analytical techniques that have been available for many years are still not used routinely in

residue analysis. Mass spectrometry is used extensively for identification and confirmation of residue identity, but it has not been adapted to routine

analysis because of its cost and the degree of expertise required to maintain the system. More routine use of mass spectrometry is expected in the future, however.

Portions of methods can be routinely automated.

Equipment that is manufactured with microprocessor control units, such as automated injectors for

chromatography, is one example. The likelihood

that complete methods will be automated within the

next 10 years is small because of the diverse sample types that are encountered and the individual

challenges that each poses.

A commonly acknowledged disadvantage of existing multiresidue methods is their “macro” design, which is based on analysis of a 100 g portion

of sample. This analytical portion is larger than

those used in recently developed methods and results in increased analytical expense from greater

volumes of solvents required. This macro scale approach was initially validated with the MOG procedure and subsequently copied in other methods

to assure that the size of the analytical portion would

be representative of the amount of food collected

(10-20 lbs.). A current FDA study is statistically addressing analytical sample size and homogeneity

issues to establish a basis for reduction in sample

and solvent volumes. Findings of this study should

have a major impact on future method development

efforts as well as future usage of current methods

in “scaled down” versions. The ultimate goal is to

achieve more rapid and efficient methodology without sacrificing analytical integrity.

The cleanup step is often a limitation in residue

methods because it generally consumes a large

amount of the total analysis time and restricts the

number of pesticides that are recovered. Development of new, more effective or efficient approaches

to removing unwanted materials in sample extracts,

while minimizing the restrictions on number of

residues recovered, would significantly improve

analytical capability. Automation of cleanup procedures offers a partial solution in that it frees the

analyst for other tasks. Application of automated

cleanup procedures is itself severely limited however, since efficient use of automation requires that

a large number of predictable analyses be planned

for similar samples. As noted throughout this paper,

development of determinative procedures that can
120

tolerate extracts with less stringent cleanups will

be a dominant factor in considering the cleanup

issue.

Most of the above focuses on enhancement and

adaptation of the type of methodology most widely

employed in residue monitoring. Screening methods, e.g., immunoassay methods, may provide a useful extension to residue monitoring activities in the

future. Although the concept of screening is not

new, screening is defined and used in a number of

different ways by regulatory agencies and others.

One type of screening is aimed at providing rapid

“yes/no” answers for one or more selected residues

at specified levels, usually levels of regulatory interest in a compliance situation. A positive result

would trigger reanalysis by more conventional and

time-consuming quantitative methods. Although

this screening would permit analyses of more samples, the time savings could be reduced or eliminated if followup analyses had to be conducted on

a large proportion of the samples. The real gain in

efficiency will thus need to be considered before

screening analyses are applied.

Coverage for certain selected residues might increase with addition of screening methods. However, designing the monitoring program to incorporate these methods will require careful planning.

The need to be able to summarize and evaluate data

from diverse methods will remain a dominant

factor.

Because residue analysis is so challenging and its

successful application relies so heavily on the expertise of the analyst, development of new personnel is of critical importance to FDA. Within the next

10 years, the majority of today’s FDA pesticide experts will have become eligible for retirement; recruitment and training of their replacements are

vital considerations to the agency.

Impacts of Laws and Regulations

The laws and regulations governing the use of pesticides on foods in the United States have had a necessary impact on the development of the analytical

methods used to enforce those laws. In turn, the

capabilities and limitations of the methods have

sometimes indirectly caused changes to be made

in the regulations.

Two amendments to the Food, Drug and Cosmetics Act originally provided the basis on which

the requirements for pesticide residue analytical

methods depend: the Miller Pesticide Residue Amendment of 1954 and Food Additives Amendment of

1958, These laws established the concept of tolerances to describe the maximum residue limits of

individual chemicals that would be permitted on

specified foods. These limits in turn established the

analyte concentration levels at which analytical

methods would be required to function reliably.

The practical imperative for multiresidue analytical methods was also provided by these two amendments because they permitted more than one pesticide on a single food commodity. (Unknown spray

histories for most foods and inadvertent pesticide

contamination of nontarget foods provide other reasons for the reliance on multiresidue methods,)

Early laws established zero tolerances for certain

pesticides in certain commodities. The abandonment of this concept was dictated by advancements

in analytical methodology which permitted determination of ever-diminishing quantities of residue and made the zero tolerance concept impractical. In a similar way, practical analytical capabilities

are taken into account when reducing tolerances

or action levels for pesticides whose uses have been

suspended, and in setting action levels for unavoidable contamination from environmental sources.

In actual practice, FDA’s analytical methods are

applied at limits of quantitation sufficiently below

the tolerance levels to provide data on incidence

and levels of residues (both above and below tolerances) in the food supply, while still being realistic

in terms of the effort required for each analysis,

These data are vital for evaluation of pesticide regulations. Typical examples are the following: (1)

FDA data for DDT findings from 1964 to 1969 were

used in 1970 to reassess tolerances and resulted in

cancellation of registration for certain uses and

lower tolerances for other uses; (2) FDA’s historic

findings of aldrin and dieldrin were pertinent in

the cancellation hearings of these pesticides; (3)

FDA’s lindane findings from 1964 to 1980 were used

by EPA in its Special Review (56) to calculate the

changing dietary exposure to lindane residues; (4)

FDA data on methomyl residues from 1978 to 1987

are being used by EPA in its tolerance reassessment

for re-registration of this pesticide; and (5) FDA data

are used to assist in setting action levels for a pesticide when its use is suspended and the corresponding tolerance is no longer applicable.

FDA’s monitoring program also directs agency

laboratories to maintain uniform limits of quantitation below tolerance levels since levels for a given

pesticide are not the same for all commodities. Tolerances for permethrin, chlorpyrifos and dimethoate in apples and peaches illustrate this complexity. Tolerances for these pesticides on apples are

0.05, 1.5, and 2.0 ppm. respectively. The correspond-
ing tolerances on peaches are 5,0, 0.05, and none

since dimethoate is not registered for use on peaches.

Analytical procedures for both sample types are

identical and cannot be readily adjusted for a given

tolerance/commodity combination. In most cases,

such adjustment would not result in significant savings in analytical cost or time.

EPA regulations have long required that registrants provide an enforcement method for each tolerance being requested. As mentioned earlier, these

methods constitute the bulk of PAM II. Since PAM

II is the reference of first choice when a single residue method is needed, it is important that the methods be reliable. Registrants must be encouraged to

adhere to the spirit of this requirement and provide

methods usable by regulatory laboratories without

excessive adaptation,

A more recent EPA requirement (56) has permitted the expansion of FDA multiresidue methods to newly introduced pesticides. Registrants

must determine analytical behavior of a new pesticide through these methods. This additional information provides FDA, state governments, and the

food industry with better tools to inform consumers

about pesticide residues in food. Availability of this

information also frees research resources of these

institutions to concentrate on development of methods for the more difficult compounds.

Certain recent situations have demonstrated that

establishment of different acceptable residue levels

by different government bodies have a profound impact on regulatory decisions, which in turn affect

the development and application of residue methodology. The international organization, Codex, is

seeking to remedy the international level of this

dilemma by proposing pesticide maximum residue

limits for adoption by member countries. This effort is expected to become more important as the

level of international trade increases.

The Food and Agriculture Organization (FAO) of

the United Nations and the World Health Organization (WHO) cooperate in a program that collects information from 34 countries on levels of pollutants

in foods and other environmental samples. This

Food Contamination Monitoring Programme is designed to assess human exposure and allow estimates of health threats caused by such pollutants.

One of the main objectives of this portion of the

Global Environment Monitoring System is to provide Codex with levels of pesticide residues in food

to assist that organization in its determination of

maximum residue limits. FDA’s data base of quantitative residue data has permitted the United States

to contribute requested data to this program throughout its history.

121

1.

2.

3.

4.

5.

6.

7,

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11,

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123

Pesticide Design: Outlook for the Future

Jack R. Plimmer, and Kenneth R. Hill, Environmental Chemistry Laboratory, Natural Resources Institute

Julius J. Menn, Plant Sciences Institute, Agricultural Research Center, Agricultural Research

Service, U.S. Department of Agriculture, Beltsville,

Content s

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Economics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Screening .. .. .. .. .$. .$. .O . . . . . . . . . . . . . . . . . .

Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Design of New pesticides . . . . . . . . . . . . . . . . . . . . . .

MD20705 -

Page

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 123

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 124

. . . . . . . . . . . . . . . . . . . . . . . . . . . , 125

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

,.0.., . . . . . . . . . . . . . . . . . . . . . . 126

,..,,. . . . . . . . . . . . . . . . . . . . . . . 127

Emerging Classes of Compounds: Examples of Specific Analytical Problems .. ....129

The Utility of Pesticide/Pest Chemical Interactions . ...........................131

Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................132

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........134

Figures

Figure Page

I. Structures of Representative Synthetic Pyrethroids . .........................126

2. Biphenylmethylpyrethroid Series (Plumber) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..128

3. Sulfonyl Ureas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....129

4. Separation of Pyrethroids on GLC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......131

Tables

Table Page

l. Ten Most Widely Used Herbicides and Insecticides in the USA . . . . . . . . .......125

2.The Quantitative Structure Activity Relationships of Some Benzoyl Phenyl Urea

Larvicides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...128

3. Recommended Application Rates of Selected Pyrethroids. . ...................130

4. Relative Mammalian Toxicities of Selected Pyrethroids . .,,,,,..,,,,,..,...,,13 1

The need to analyze pesticide residues in food and

drinking water for regulatory purposes creates increasingly complex analytical problems because so

many diverse molecular types must be determined

in a large variety of crops or foods. Multiresidue

procedures (MRPs) are important because a method

must detect as many pesticides as possible and it

must be applicable to samples of unknown treatment history.

For a number of reasons, including the high costs 

of development and safety tests, reduced success

in screening programs, and pest resistance, the

number of pesticides entering the U.S. market has

decreased in recent years. However, there continues to be a strong demand for pesticides, partitularly herbicides, and this is likely to continue

into the future. Despite rapid progress in fundamental aspects of biotechnology, its widespread application to pest control technology will proceed at

a steady pace because many questions of safety must

be answered.

New biochemical and biological knowledge is important in developing new leads for synthesis, and

quantitative structure activity relationships are

guiding the optimization of promising, active

molecules, Complexity may increase as products

of microbial metabolism (such as the avermectins)

are found to be pest control agents.
124

Extremely low rates of application result in low

residue levels, and the detection and quantitation

of such low levels presents a considerable challenge

to analytical chemists and designers of instrumentation. The sulfonylureas and the pyrethroids are

two examples of classes of pesticides that may be

used at very low rates of application and, in consequence, require analytical methods capable of determining residues at the parts-per-billion level.

Novel methods of analysis (such as biosensors)

are being developed, and immunoassay techniques

are increasing their range of applicability and sensitivity. The latter are beginning to meet the need

for simple and rapid screening procedures that may

simplify the task of the analyst.

A consequence of the increased complexity and

the potent biological activity of new pesticide

molecules is the need for more sensitive methods

of detection and improved methods of separation.

Multiresidue methods will continue to be adaptable to many new compounds. It is recommended

that information concerning their applicability to

new compounds be made readily available.

Introduction

There is increasing awareness among consumers

of the potential of man-made chemicals to contaminate sources of food and drinking water. There is

particular concern over the implications of food

contamination by pesticide residues. The capability of analytical techniques to detect extremely low

levels of trace contaminants has continually expanded. However, knowledge of the toxicological

significance of such contaminants has not progressed

at the same rate. It is also important to bear in mind

that the study of the effects of individual compounds

on biological organisms does not provide satisfactory information concerning the biological effects

of several interacting compounds.

Residue analysis may be conducted for several

purposes. The registration and use of a pesticide

is preceded by supervised trials to determine the

rate of disappearance. Residues on raw agricultural

commodities must also be determined.

For regulatory and monitoring purposes, residues

in food for human consumption and residues in

environmental samples must be determined in samples that do not have a known treatment history.

Therefore, procedures must be employed that can

detect as many pesticides as possible in the most

economical way. Multiresidue procedures (MRPs)

are used for this purpose, and these are usually

limited to the parent compound and closely related

compounds. An excellent survey of the scope and

capabilities of MRPs appeared in a recent International Union of Pure and Applied Chemistry report

(3).

Rapid developments in analytical technology contribute to, but cannot be equated with, improved

ability to determine the presence and amounts of

contaminants in food. Much progress has been

limited to the analysis of specific analytes or groups

of analytes. Before beginning an overview of developments in agricultural chemicals, it should be

stressed that the problem of analysis requires for

its solution that we consider both analyte and matrix. The former is the compound of interest, a definition that is often extended to cover not only the

parent compound but also its metabolizes and transformation products; the latter refers to the particular crop or food type for which the information is

desired.

The magnitude of this problem can be gauged by

considering the efforts of the Codex Alimentarius

Committee on Pesticide Residues (CCPR), which

has established minimum residue levels for 150

compounds and more than 2,500 pesticide/commodity combinations over a period of 20 years (10),

Multiresidue procedures are essential if it is necessary to determine as many pesticides as possible

in various types of matrices. The complexity of the

problem will increase in future years as new classes

and types of pest control agents are introduced in

response to a variety of constraints.

Economlcs

There is little likelihood that agricultural production and pest control will abandon their prime reliance on chemical methods of pest control in the

coming decades, although there will be greater emphasis on the use of biological controls and technology that will contribute to the reduction of pesticide use. The market for agrochemicals continues

to grow but more slowly than in the past. In the

United States, there was an 8 percent decline in

cropland from 1986 to 1987, and it was predicted

that pesticide use would decrease 9 percent during

that period (30). A market study predicted that herbicide growth would be most rapid primarily for

corn and soybeans with a growth of about 5.3 percent per annum to a value of $3.47 billion. Expansion would emphasize new compounds (such as the

imidazolinone and sulfonyl urea herbicides) that

possess new modes of action and are used at extremely low rates. The synthetic pyrethroids that

now account for about one-third of world insecti-
125

cide use (27) would lead the way in insect control,

and new fungicides, primarily the ergosterol biosynthesis inhibitors, would be emphasized, Table

1 indicates the herbicides and insecticides that are

currently most widely used in the United States.

Economics of pesticide production is a major factor in change. The end of patent protection for a

number of compounds of major importance means

that there will be a shift to a commodity market with

a more competitive approach. Pesticide manufacture is also becoming consolidated. Foreign buyers

have now acquired many U.S. businesses. The agrochemical market is international, but the developing countries’ market has not materialized to the

extent anticipated. New compounds are slow to appear on the market. Successful introductions have

dropped from 60 new compounds between the

1950s and ’60s to 21 between the 1970s and ‘80s.

Because the costs of research, development, and

market introduction have increased to about $40

million per compound according to data developed

in 1982 (22), the market will be largely restricted

to major international companies who will emphasize the needs of major world crop markets. Profitability continues to be limited by the time that

elapses between discovery, market introduction,

and patent expiration. About 13 years is needed to

reach a break-even point after discovery.

The food producer must also cope with major constraints because the cost of pesticide development

is passed on unaccompanied by any increase in

farm prices. Thus, the farmer must continually review the cost of all chemical inputs to adjust pesticide and fertilizer use to maximize his return. To

attain this goal in part may be practicable if substantially lower rates of application can be achieved

by using pesticides of greater biological effectiveness and by using better application technology,

Screening

Although there is little doubt that synthetic chemical pesticides will continue to be the main weapon

in our crop protection arsenal in the next 10 to 20

years, the rate of new compound introductions has

dramatically decreased in recent years. This decrease is largely due to the reduced number of companies engaged in agrochemical research and development, to the difficulty in discovering viable

new pesticides by the process of empirical synthesis and screening, and to cost and safety considerations.

The rate of commercial success from screening

to market development has fallen from 1 in 1,800

in 1956 to 1 in 15,000 in 1979 (21), and this adverse

ratio is expected to increase in the coming years.

From the 1950s through the 1970s, the majority of

insecticides were neurotoxicants represented by

chlorinated hydrocarbons, carbamates, and organophosphorus esters. These pesticides have similar

modes of action in insects and vertebrate species,

including humans. Today, the largest class of insecticides in use are the synthetic pyrethroids,

which are also neurotoxicants. Representatives of

this class are shown in figure 1.

At present, the major agrochemical companies are

directing greater efforts and resources toward more

fundamental research to discover novel classes of

pesticides. Morrod (23) discussed approaches to current and future directions for discovery involving the

following: novel synthesis, speculative biological

chemistry, directed synthesis, natural product anaTable 1 .—Ten Most Widely Used Herbicides and Insecticides in the USA

Herbicides Insecticides

Common name Trade name Common name Trade name

alachlor Lasso aldicarb Temik

atrazine Aatrex carbaryl Sevin

butylate Sutan carbofuran Furadan

cyanazine Bladex chlorpyrifos Dursban

2,4-D many malathion Cythion

glyphosate Roundup methyl parathion Penncap

metolachlor Dual parathion Folidol

metribuzin Sencor phorate Thimet

propanil Stare synthetic pyrethroids many

trifluralin Treflan terbufos Counter

SOURCE: P.C Kearney, A R. Isensee, and J.R. Plimmer, “Contribution of Agricultural Pesticides to Worldwide Chemical Distn.

but(on, ” Toxic Corrfarn/rratiorr in Large Lakes, vol. Ill, N.W, Schmidke, Lewis, Chelsea, Ml, 1988.
126

Figure I.-Structures of Representative

Synthetic Pyrethroids

cl \

CN

SOURCE: C R Wotihing ed, %dcti Manuel, 8th edtion, Brttish Crop Protection

Caned, Thornton Heath, UK, 1987

log synthesis, and greater reliance on quantitative

structure-activity relationship (QSAR) methods.

Safety

An important part of the high cost of a pesticide

is the continual increase in the cost of safety tests.

Environmental consequences and health effects of

pesticide use continue to be major topics of public

and regulatory concern. Testing for acute and latent toxicity is a substantial portion of the cost of

pesticide development. These costs and the regulatory implications of such factors as the production

of oncogenic responses in test animals substantially

influence the current directions of chemical innovation. A recent National Academy of Sciences

study on the issue of pesticide residues in food has

addressed some controversial issues involved in

pesticide regulation, especially as they pertain to

the Delaney Clause (26).

Because the environmental behavior of a pesticide is largely determined by its chemical structure,

the constraints on the selection of structural classes

continue to be more pressing. For example, the contamination of groundwater by pesticides may result from agricultural use under certain conditions

depending on soil, crop, method of application, etc.

Although the amounts reaching groundwater may

be well below the limits deemed as potentially harmful to human health, the fact that such trace amounts

are present was not predicted on the basis of existing knowledge at the time of registration. Aldicarb,

alachlor and atrazine are among the compounds detected in groundwater, and a number of survey programs are planned or in progress to determine the

scope of the problem.

Studies are being undertaken to detect precise

conditions under which contamination occurs and

to limit its occurrence by changes in pest management practice. However, the recognition that the

major contributing factors are the soil environment

(e.g., soil fractives; channels; agricultural practices;

microbial activity; moisture; clay, mineral, and organic matter content; etc.) and the structure and

physical properties of the pesticide molecule (e.g.,

rate of degradation in soil, water volubility, vapor

pressure, partition coefficient between water and

soil, organic matter, etc.) lead to the conclusion that

pesticide design must take into account leachability or the potential for biologically active materials

to move vertically in soil to groundwater.

The regulatory foundation for safety issues will

continue as a major factor in the design of chemicals. The emerging issues include the U.S. Environmental Protection Agency (EPA) actions to bring

the Federal Insecticide, Fungicide and Rodenticide

Act (FIFRA) into compliance with the Endangered

Species Act in the United States, beginning in 1988.

This action will prohibit pesticide use in specified

areas that are the habitats of endangered species.

The issues of health and safety are not only of concern to the consumer. Farmers, formulators, applicators, and field workers are by their occupations

exposed to pesticides. Home and garden use is also

an important safety consideration. Thus, the scope

of regulation extends over a wide range of activities.

Thus, only a limited number of structural types

will be considered for development as they emerge

from the elaborate program of safety testing. Analytical considerations will follow these dictates in

so far as they are part of the accountability needs

during the process.

Resistance

A further constraint on molecular design is the

problem of pest resistance to pesticides. Insecticides

are particularly susceptible, and reliance on chemical classes that possess closely related modes of

action or similar target sites hastens the obsolescence of particular classes of compounds. The response of the manufacturer is to broaden the chemical screen to search for new modes of action, and

several classes of insecticides and herbicides introduced in recent years reflect this consideration. As
127

an example, diflubenzuron may be considered the

first of the commercially introduced chitin synthesis inhibitors. Chitin, the skeletal material of insects,

is absent in man and vertebrates. Innovative pesticide design and the search for alternatives will continue to challenge the potential development of resistance,

In summary, many factors have combined to create pressure to reduce the use of pesticides in food

production. These include economic and regulatory

pressures at the producer and farm level. In addition, changes in agricultural management (for example, adoption of conservation tillage, change in

land use, and new irrigation and application systems) and formulations have contributed to the evolution of new agrochemicals. Although biological

control and developments in biotechnology will

contribute to decreased reliance on chemicals, it

is probable that at least a decade will elapse before

a substantial contribution can be expected from

such sources. These changes will then be accompanied by shifts in regulatory emphasis. The current problem of analysis of pesticides may be relatively less complex than the problem of satisfying

the safety concerns arising from new technologies.

Design of New Pesticides

As discussed previously, more rational approaches

are needed to improve the odds for developing a

successful, marketable pesticide chemical.

In recent years, there have been considerable advances in the understanding of basic biology, biochemistry, and the physiology of host and target species. In addition, there is improved understanding

about the site of action and effects of pesticides at

the molecular, cellular, and whole organism levels.

These have contributed substantially to rationalization of approaches to molecular design of pesticides. Research discoveries as related to bioactivity

have been greatly aided by the development of

regression functions that form the basis of QSAR.

QSAR combines elements of quantum chemistry,

biodata, and computerization to fit parameters

predefined by biochemical processes. Knowledge

derived from this methodology should provide a better foundation for the rational design of novel,

highly active, and environmentally sound crop and

livestock protection chemicals. More detailed

aspects of QSAR in pesticide design were reported

in a symposium on this topic (17),

The following examples illustrate the utility of

QSAR in optimizing synthesis and bioactivity.

Nakagawa et al, (24) described the optimization

of quantitative structure-activity of benzoylphenylurea

larvicides with reference to substituents at the aniline moiety against the major rice insect pest, the

rice stem borer (Chilo suppressalis Walker).

Table 2 shows the empirical formula for a series

of N-2,6-difluoro and N-2,6-dichlorobenzoyl-N’-(4-

substituted phenyl)ureas and the regression equation parameters used in the QSAR analysis to predict optimal insect (chitin synthesis inhibition) activity. The analysis was performed with each compound

synergized with piperonyl butoxide (PB) to reduce

metabolic degradative effects in the insect.

Also included in table 2 is the resultant analysis

for four compounds in the series. Activity is enhanced by electron withdrawing (op) and hydrophobic substituents ( ) and reduced by bulky groups

(AB).

Plummer (28) succeeded in designing a novel series of highly active biphenylmethylpyrethroids

through the QSAR approach. His success was specially significant since it came when the field appeared to be already saturated with synthetic

pyrethroids.

From these studies, Plummer concluded that

where X = F or CH3, activity was optimal resulting

from the confirmational preference of these compounds for a twist angle at about 500 involving ring

B. The latter is most likely involved as a ligand of

the active site, involving a specific fit (figure 2).

In a comprehensive QSAR study of terpenoid and

non-terpenoid insect juvenile hormone mimetic

compounds (juvenoids), Nakagawa et al. (24), through

regression analysis and correlation equations formulated for 85 compounds on two insect species,

developed hypothetical “mode of action” models

involving overall similarity as well as species differences at the receptor site showing structural conditions necessary for activity. Without such quantitative calculations, it would have been difficult to

predict similarity in the mode of action of such diverse compounds as terpenoids and N-alkyl-N,Nethylenebis (thiocarbamates).

The QSAR approach to design of candidate compounds offers a great deal to the analytical chemist

who shares the need for much of the physiochemical data, such as the octanol/water partition coefficients that must be generated for the calculation of

regression functions. Analytical schemes could benefit by close cooperation at the pesticide design

stage.

Undoubtedly, greater structural diversity is in

store for the future as biochemically inspired targets in insects, weeds, and fungi are better understood and exploited.

Such new bioactive models include insect neuropeptides (15, 22), which provide potential new

vistas in insect control by which insects’ native
128

Table 2.e Quantitative Structure Activity Relationahips of Some Benzoyl Phenyl

Urea Larvicides

1A Cl 6.32 6.64 0.23 0.60 0.49 0.79

IIB CF 6.86 6.92 0.54 1.60 0.93 1.42

Ill CH3 5.10 4.60 -0.17 1.04 -0.02 -0.03

IV CH3 3.47 4.30 -0.27 2.07 -0.41 -0.73

Y=2, 6-D (ly=O). A. Diflubenzuron B. Penfluron

Larvicidal activity of piperonyi butoxide (PB) synergized compound against larvae of rice stem borer

(C/vlo suppn?ssa/k Walker)

SOURCE: Nakagawa et al., “CJuantltative Structure-ActMy Studies of Benzoylphenylurea Larwcldes,” %s#c. Bhxhawn. & Physid.,

21:309-325. 1984

Figure 2.- Biphenylmethylpyrethroid Series (Plummer) biochemical serve as prototypes for lethal peptide

agonists and antagonists (19).

It is likely that in the next two decades, increasing resources will be directed toward pest management technologies that involve the use of microbi-

\

ologicals, natural products, genetic and behavioral

biochemical, and transgenic plants (20).

/

c

x

The question of the impact of biotechnology on

CH3

pest control presents difficulties because the future

direction of expansion is not clear and techniques

X= F or CH3 are in the exploratory stage at present. For examSOURCE: C R Worthing ad, %sUcti Mewe/, 8th edition, Brttish Crop Protection

pie, a technique that appears promising is the poCouncd, Thornton Heath, UK, 19137 tential control of insects that attack corn roots by
729

infecting corn with a vascular, endophytic microorganism that carries the gene capable of expressing

the bacillus thuringiensis endotoxin. This technique

and some others that rely on gene transfer to plants

or microorganisms depend on the expression of

toxins to achieve insect control. Safety considerations in biotechnology are viewed quite differently

from considerations of food contamination by

residues of synthetic pesticides, although some of

the same questions must ultimately be asked, and

it seems likely that the analysis of bacterial toxins

or other complex molecules of biological origin will

become more important in future. Because MRPs

exclude biological macromolecules during the cleanup

stage, methods of study or assay that have been developed for biological or clinical studies will probably be more appropriate in this field and immunoassay would appear to be a logical technique.

These new developments will pose increasingly

more difficult challenges to the analytical chemist

in the quest of accessible and practical residue analytical methods.

Emerging Classes of Compounds:

Examples of Specific Analytical

Problems

A number of newer herbicides are active at extremely low rates of application. For example, fluazifop applied at 4 to 8 oz. per acre will control annual grasses and perennial weeds. Sethoxydim is

active at 3 to 7.5 oz. per acre, chlorsulfuron at 0.17

to 0.5 oz. per acre, and chlormeturon at 1 to 5 oz.

per acre.

Several manufacturers are involved in the development of these compounds. Chlorsulfuron and sulfometuron (figure 3) are the active ingredients of

GleanR and OustR, respectively, both introduced

by DuPont. Other manufacturers have introduced

similar herbicides. The activity of these and other

new herbicides currently being developed is extremely high, and as application rates will be low,

the residue levels in soils and plants will also be

extremely low.

Residue levels in agricultural products will be so

low as to challenge the ingenuity of the analyst.

Since chlorsulfuron and sulfometuron are both thermally unstable, they cannot be directly determined

by GLC. Chlorsulfuron was analyzed by gas chromatography after conversion to the methyl derivative. Residues in agricultural runoff water were determined at the parts-per-trillion level (z). The

earliest method for analysis in soil at the parts-perbillion level relied on HPLC separation combined

Sulfometuron

Metsulfuron

Triasulfuron

(proposed)

Primisulfuron

(proposed)

Figure 3.- Sulfonyl Ureas

,CH3

—

‘CH3

,OCH3

—

C H3

I

OCH3

SOURCE: C R Worthmg ed , l%sstiide Menua/, 8th edtion, Brltwh Crop ProtectIon

Councd, Thornton Heath, UK, 1987

with photoconductivity detection. Because extraction procedures normally used for soil liberate quantities of ultraviolet-absorbing material, there is considerable interference with the operation of the UV

detector (33). The procedure was used because no

chemical methods were available when field evaluation was conducted. A 5-day incubation period

gave the most satisfactory data. Groves and Foster

(11) described a bioassay for chlorsulfuron in soils

that was based on the inhibition of corn root growth

after a 7-day period of development in soil containing chlorsulfuron. The benefit of such bioassays is

their reliance on simple techniques and their potential accuracy, For such highly active herbicides,

simple bioassays may offer some advantages. Disadvantages are the length of time needed to conduct the bioassay and the need to conduct the test

in a greenhouse or growth chamber.

Chlorsulfuron is a water-soluble compound, and

a scheme for extraction and separation of the compound and its metabolizes from treated plants was

proposed by Bestman et al. (5) using aqueous ex-
130

traction of plant tissue. Subsequent chromatography on a reverse-phase column and elution with

aqueous formic acid/methanol gave an average recovery of 94 percent (based on 14C data). The use

of reverse-phase solid-phase extraction for analysis of aqueous environmental samples has been advocated as a general method for trace organics, and

this appears to work well in the case of the sulfonylurea herbicides (31). The paper contains useful suggestions for the development of procedures

for a solid-phase extraction method and discussion

of the potential value of this technology for extracting of organic compounds from aqueous solutions.

Confirmatory procedures for the identification of

sulfonylureas include combinations of gas chromatography with liquid chromatography (29, 18).

An immunoassay analysis for chlorsulfuron can be

used to determine chlorsulfuron in unfiltered soil

samples at nanogram levels (16) and the technique

appears promising. The authors comment that the

method is relatively specific in contrast to the bioassay method, and interferences with the HPLC

method may raise detection limits considerably.

Analytical methods for the new herbicides are

thus in an evolutionary stage. The extremely low

levels at which residues are to be expected contribute to the analytical problem, but these low levels

represent a desirable factor in future pesticide

design.

Bioassay is useful for the determination of chlorsulfuron, as well as for dichlofop acid and sethoxydim residues in soil at very low levels. The test

involves measurement of the root length of pregerminated oat or corn seedlings (13).

An example of the trend in insecticide development can be found in the class of compounds known

as synthetic pyrethroids. The synthetic pyrethroids

are derived from the structures of natural pyrethrins,

a series of chrysanthemic acid esters extracted from

chrysanthemum flowers. Beginning with allethrin

in 1949, both the acid and alcohol moieties of the

ester have been replaced, modified, or substituted

to produce a family of insecticides having greatly

enhanced activity and stability. The original

pyrethroids could only be used indoors as sprays

in homes and greenhouses due to short residual

activity. However, the discovery that halogenation

of the vinyl moiety of the chrysanthemic acid increased photostability and enhanced insecticidal

activity led to the modern pyrethroids that can be

used as field insecticides on crops.

The application rates of some of the current products are measured in grams/acre instead of the traditional pounds/acre of other pesticides (table 3). RelaTable 3.—Recommended Application Rates of

Selected Pyrethroids

Fenpropathrin decreasing

Compound

Permethrin

Fenvalerate

Fluvalinate

Flucythrinate

Cypermethrin

Tralomethrin

Cycloprothrin

Cyfluthrin

Deltamethrin

Alphamethrin

Karate

Phenothrin

Fenpropathrin

Other insecticides for comparison:

Chlordane OC

Aldicarb carbamate

Carbaryl carbamate

Malathion O P

Diazinon OP

Chlorpyrifos OP

Parathion O P

Diflubenzuron IGR

Rate. lb./Acre

0.050-0.200

0.050-0.200

0.025-0.100

0.025-0.080

0.020-0.075

0.013-0.024

0.009-0.180

0.009-0.045

0.008-0.024

0.0045-0.027

0.0045-0.027

0.004-0.016

0.002-0,010

1.0-10.0

0.5-10.0

0.5-4.0

0.5-3.0

0.25-2.0

0.10-5.0

0.10-1.0

0.02-0.14

SOURCE: Agricultural Chemicals, Book 1, Insecticides, W.T. Thomson, (cd.)

(Fresno, CA: Thomson Publications, 19S6).

tive mammalian toxicities are shown in table 4. The

lower application rates of the synthetic pyrethroids

are due to their greater toxicity to insects, but not

to mammals. For example, permethrin and carbaryl

have about the same mammalian toxicity, but permethrin can be used at rates about 10 to 20 times

lower than carbaryl (tables 3 and 4). Similarly, deltamethrin and chlorpyrifos have similar mammalian

toxicities, but deltamethrin rates average about 100

times less than chlorpyrifos. Therefore these lower

application rates also imply that the potential health

hazard is reduced. Low application rates and consequent low residues and the lipophilicity imparted

by the halogen functional groups determine the approaches used in developing multiresidue methods

of analysis. Residues can be extracted by methods

already developed for the organochlorine insecticides such as DDT, etc. Fortunately, the group is

characterized by fairly high melting and/or boiling

points, which permit their separation from other

halogenated compounds by high-temperature gas

chromatography and sensitive electron-capture detection. The lipophilic properties also result in accumulation in animal fat when treated grains, forage, and other crop products are fed to animals.

However, the general structure of this family of

compounds results in both cis/trans isomers and optical isomers, which complicate the chromatographic
131

Table 4.—Relative Mammalian Toxicities of

Selected Pyrethroids

Increasing Toxicity

LD (rat, oral), mg/kg

Compound body wt.

Phenothrin

Cycloprothrin

Tralomethrin

Cyfluthrin

Fenvalerate

Permethrin

Fluvalinate

Cypermethrin

Deltamethrin

Alphamethrin

Flucythrinate

Karate

Fenpropathrin

Other insecticides

Diflubenzuron

Malathion

Carbaryl

Diazinon

Chlordane

Chlorpyrifos

Dichlorvos

Parathion

Aldicarb

for comparison:

Class

IGR

OP

carbamate

OP

OC

OP

OP

OP

carbamate

10,000

5,000

1,070

500

451

450

261

200

128

79

67

56

54

4640

1375

500

300

250

135

56

3

0.79

SOURCE: Agricultural Chemicals, Book 1, Insecticides, W.T. Thomson, (cd.)

(Fresno, CA Thomson Publications, 1986).

determination step. If resolution of the isomers is

desired, then high-quality capillary column GC must

be used. Some success along this line has already

been achieved with special large-bore capillary

columns as demonstrated by a typical gas chromatogram showing the separation of four pyrethroids

in a fortified animal-fat extract (12). The success of

the pyrethroids as agricultural insecticides will

likely lead to new structural variations in the future with even more enhanced stability and activity (figure 4).

The Utility Of Pesticide/Pest

Chemical interactions

Many pesticides act by inhibition of an important enzyme system. In those cases where the mode

of action is well defined and the activity of the enzyme can easily be determined, a method of analysis based on enzyme inhibition may be very useful

as a screening technique. For example, organophosphate esters or carbamates inhibit the enzyme

cholinesterase, which is responsible for the hydrolysis of the neurotransmitter acetylcholine. Rapid

assays have been based on calorimetry or radioacFigure 4.–Separation of Pyrethroids on GLC

SOURCE: Hill, Kenneth, Agriculture Research Service, U S. Department of Agriculture, BeHsville, MD.

tivity as a measurement of the extent of reaction.

For assay, acetylcholine chloride is used as a substrate to determine the activity of cholinesterase in

a sample (blood, tissue, etc.) The reaction produces

acetic acid, which can be detected by an indicator

dye or, using 14C-acetylcholine chloride, by determining residual radioactivity in the sample after

removing acetic acid by evaporation. Indicator

papers are commercially available for field tests of

insecticides that inhibit cholinesterase. Such tests

are useful for screening and indicate the presence

of one or more compounds of the general class, Although other types of enzyme inhibition may be

common to classes of pesticides, and methods of

analysis based on these reactions are feasible, they

have not been widely exploited or passed into regular analytical use for pesticide determination.

A method of analysis for chemicals affecting insect behavior involves the detection of pheromones

by isolated insect antennae. Since this method offers

unique selectivity, it has been used as the basis of

gas chromatographic detection (4). The method depends on specific recognition of a complex organic

compound by a biological receptor site. Recognition of an organic molecule by a specific receptor

is also the basis for the immunoassay techniques,

which depend on the interaction between a pesticide and a complex antibody. The production of antibodies capable of recognizing individual pesticides or groups of pesticides is being rapidly

exploited, and immunoassay techniques are currently available for qualitative analysis and quantitative determination of pesticides. The ability to

recognize a class of pesticides renders this tech-
132

nique extremely suitable for screening. Its advantage is that it relies on some degree of correspondence between the biological site (on the antibody)

and the pesticide, whereas many enzyme systems

function in situ and the site of action of a pesticide

in a linked series of processes may be difficult to

define or isolate for use as a basis for an analytical

technique.

A further example of a system that may be useful

for screening purposes is the ability of many herbicides to inhibit photosynthesis. This activity may

be correlated with inhibition of the ability of cellfree plant extracts to catalyze a light-dependent evolution of oxygen in the presence of an acceptor such

as ferricyanide, a process known as the Hill reaction

(8). A variety of herbicides inhibit the Hill reaction

(ureas, triazines, uracils, dinitrophenols,

diphenylethers, pyrimidones, carbamates, anilides,

etc.), and such a reaction may have analytical utility as a screening tool.

Pesticide/pest chemical reactions may be useful

in the future as part of a screening system to indicate the presence or absence of one or more of group

of analytes. A procedure that demonstrates the presence of one or more of a very wide range of compounds could provide a useful screen to indicate

which samples should be selected for further analysis.

The value of cholinesterase inhibition as a rapid

field method is well accepted, but new approaches

are needed to combine biochemical and analytical

thinking in devising procedures that will provide

potential for both broad screening and quantitative,

specific detection of analytes. There is an indication that some biosensor techniques can meet the

latter need, but at present, biosensors are primarily developed to address specific problems of substrate analysis. A sensor that could respond to each

individual member of a group of analytes still remains beyond practical limits.

Methods based on biological properties (immunoassay and enzyme inhibition) are likely to find application in rapid screening of samples in order to

eliminate negative samples prior to instrumental

analysis in a laboratory. Biological methods will be

unlikely to provide satisfactory multi residue methods for the following reasons: 1) They are not sufficiently selective to distinguish members of family

groups (enzymatic methods); or 2) They maybe too

selective (immunoassay) and therefore will require

a separate reagent for each of the thousands of possible pesticides, degradation products, and metabolites. However, highly automated procedures would

permit rapid screening for perhaps a few hundred

selected compounds.

Conclusions

A number of major concerns have dominated the

design of new pest-control chemicals. Predominant

among these is the combination of optimized biological activity against target species with minimal

acute or latent toxicity toward other organisms. To

accomplish this and reduce the possibility that pest

resistance may rapidly render the product worthless, approaches to the discovery of pest control

chemicals now proceed with a greater understanding and regard for modes of action and metabolism.

In recent years, the major advances in techniques

by which structure-activity relationships are investigated and interpreted have also been reflected in

product chemistry, where the producers now offer

new chemicals that may be pure isomeric or optically active forms. Progression from the use of relatively crude materials containing many isomers or

related impurities (e.g., toxaphene) applied at rates

of several pounds per acre to chemicals that are

highly purified and are active at a few ounces per

acre has significant impact on the work of the analyst because, in the future, residues from field use

will generally be much lower.

Initially, the major problem for the regulatory analyst is the question of tolerance, and it is to be expected that reductions in rate of use will be beneficial if corresponding median lethal dose (LD50)

values remain high. Although absolute sensitivity

is not a factor in the analysis, it is to be expected

that tolerances will be reduced as methods improve.

There are many new approaches to control of

pests, and these will continue to gain ground at the

expense of chemical control. However, pest-control

chemicals are significant in an international market. Their use will continue, and there will be increasing diversity of chemical structures as molecular architecture is varied and refined to combine

biological activity with the reduction of adverse effects on nontarget organisms and the environment.

Although the range of structural types is increasing, compounds currently being developed do not

appear to present insuperable analytical difficulties.

The major problem is the increase in number and

variety and the proliferation of structures that differ only in detail (for example, the pyrethroids), thus

calling for more sophisticated separation techniques.

Multiresidue procedures appear to be adaptable

to many new chemicals, and it is now required that

the performance of new chemicals in standard

MRPs be investigated as part of the registration

process. This information is of great assistance to
4.Y Q

the regulatory analyst and it is important that it be

made readily available. However, as the number of

potential matrix/compound interactions increases,

so the difficulties of the regulatory analyst will also

increase. The parent compounds can usually be recognized by MRPs, but the type and variety of metabolic products from a single pesticide may obviate

their determination in a single MRP, or even in any

MRP. To simplify this problem, Frehse (10) has proposed that a single indicator compound should be

selected to represent the residue of a certain pesticide and its metabolizes. It was suggested that the

concentration of the indicator compound should

bear a known relationship to the concentration of

the toxicologically significant residue; in addition,

the indicator compound should be available as a

standard, be recoverable in MRPs, and sufficiently

stable for reproducible analysis. The concept of indicator compounds is a useful one and is one that

could be included in the framework of efforts to

bring about international harmonization of maximum residue limits.

The simplification of approaches to the problems

of the regulatory analytical chemist was also advocated by Frehse (10), who described a three-step system proposed by Westlake and Gunther (32). The

first stage involves screening for given constituents

to previously established limits of detectability. The

second stage consists of screening to discriminate

samples that are above tolerance from those below.

The third stage is that of quantitative analysis.

Clearly, current screening methods for groups of

compounds, such as immunoassay, are capable of

providing much information and could form part

of a tiered analytical procedure.

The major obstacle to improved multiresidue

methods is still the labor intensive extraction and

cleanup procedures required. The initial stages of

analysis involving the selection of a representative

sample, extraction, and cleanup of the extract are

critical and also time-consuming. Much more research is needed in automation and robotics to increase throughput and reduce per-sample costs for

the conventional approaches. Not much research

effort has yet been expended on techniques for eliminating cleanup steps, although direct injection of

extracts without extensive cleanup was reported as

long as 17 years ago for organophosphorus compounds (6, 7).

The introduction of synthetic organic pesticides

was followed closely by the rapid development of

gas chromatography in the early 1960s. Element

specific detectors simplified residue determination

for such compounds as the organochlorine, organophosphorus ester, and carbamate insecticides.

Procedures for extraction, solvent partitioning,

purification, and determination have evolved, but

in the past two decades there have been remarkable advances in the performance of columns for gas

or liquid chromatography. Identification of specific

residues has been made easier by combination of

liquid or gas chromatography with mass spectrometric detection. Nevertheless, a variety of compounds remain outside the capability of MRPs and

the analyst must resort to special procedures.

Highly polar or water-soluble materials often present difficulties and must be converted to lipidsoluble derivatives. Unfortunately, many metabolites belong to this category and cannot be included

in general MRP procedures. There is no simple generalization to describe new compounds appearing

on the market, and there may be difficulties if polar or thermally unstable compounds must be analyzed. Lower rates of application are to be expected

in the future because the design of biologically active molecules can more readily be optimized. If toxicity is extremely low, there may be fewer residues

of significance, but analytical needs will still exist.

Simple, rapid, and sensitive screening methods will

be essential to indicate whether further analysis of

samples should be undertaken, There is a critical

need for such methods to reduce the burden on the

regulatory analyst.

There is little reason to believe that the necessity

to continue to develop and apply MRPs will decrease in the next two decades. Agricultural chemicals will continue to be used worldwide and it is

important to protect the quality of produce reaching the consumer. However, it is important to increase effectiveness and reduce costs of current

methods and some priorities should be allocated;

among these the following may be considered:

1.

2.

Current MRPs will probably be adaptable to

many new chemicals entering the market.

However, analysis of closely related isomers

will require improved separation techniques,

and the potential requirement to determine

residues at the parts-per-billion level will demand more sensitive detectors.

Sampling, extraction, and cleanup procedures

are generally time-consuming and expensive

in terms of solvents, etc. The application of

automated techniques may avoid some labor

costs, but new technology is needed.
134

3. Rapid methods for screening that require no

processing or minimal processing of the sample would be extremely valuable, particularly

if they were applicable in the field.

4. Methodology is needed that is applicable to sep1.

2.

3.

4.

5.

6.

7,

8.

9.

10.

11.

12.

13.

aration and analysis of macromolecules or complex molecules of biological origin that might

be involved as new active principles in future

pest-control technology.

References

Agricultural Chemicals, Book I, Insecticides,

W.T. Thomson (cd.) (Fresno, CA: Thomson Publications, 1986).

Ahmad, I., “Capillary Column Gas Chromatographic Determination of Trace Residues of the

Herbicide Chlorsulfuron in Agricultural Runoff Water, ” j. Assoc. Off Anal. Chem. 70:745-

748, 1987.

Ambrus, A. and Thier, H. P., “Applications of

Multiresidue Procedures in Pesticides Residues

Analysis,” Pure and Appl. Chem. 58:1035-62,

1986.

Am, H., Stradler, E., and Rauschler, S., “The

Electroantennographic Detector—A Selective

and Sensitive Tool in the Gas Chromatographic

Analysis of Insect Pheromones,” Z. Naturforsch.

Tei~ C 30:722-725, 1975.

Bestman, H. D., Devine M. D., and Vanden Born,

W, H., “Extraction and Separation of Chlorsulfuron and Its Metabolizes from Treated Plants,”

Weed Sci. 35:22-26, 1987.

Bowman, M.C. and Beroza, M., J. Assoc. Off

AnaZ. Chem. 53:499-508, 1970.

Bowman, M. C., Beroza, M,, and Hill, K. R., ].

Assoc. Off. Anal. Chem. 54:346-358, 1971.

Corbett, J. R., The Biochemical Mode of Action

of Pesticides [New York: Academic Press, 1974).

Farm ChemicaZs Handbook (Willoughby, OH:

Meister Publishing Company, 1988).

Frehse, H., “Trends in Pesticide Residue Methodology,” Pesticide Science and Biotechnology,

R. Greenhalgh and T.R. Roberts (eds.) (Oxford,

UK: Blackwell Scientific Publications, 1983), pp.

293-300,

Groves, K.E.M. and Foster, R. K., “A Corn (Zea

mays L.) Bioassay Technique for Measuring

Chlorsulfuron Levels in Three Saskatchewan

Soils,” Weed Science 33:825-828, 1985.

Hill, K. R., personal communications, 1988.

Hsiao, A.I. and Smith, A. E,, “A Root-Bioassay

Procedure for the Redetermination of Chlorsul148

15,

16.

17.

18.

19.

20.

21.

22.

23,

24.

25.

26.

27.

furon Diclofop Acid and Sethoxydim Residues

in Soils,” Weed Research 23:231-236, 1983.

Kearney, P. C., Isensee, A. R., and Plimmer, J, R.,

“Contribution of Agricultural Pesticides to

Worldwide Chemical Distribution,” In: Toxic

Contamination in Large Lakes, vol. III, N.W.

Schmidke, Lewis, Chelsea, MI, 1988, pp. 49-60.

Keeley, L.L. and Hayes, T. K., “Speculations on

Biotechnology Applications for Insect Research,”

Insect Biochem. 17(5):639-657, 1987.

Kelley, M. M., Zahnow, E. W., Petersen, W. C., et

al., “Chlorosulfuron Determination in Soil Extracts by Enzyme Immunoassay,” j. Agric. Food

Chem. 33:962-965, 1985.

Magee, P. S., Kohn, G. K., and Menn, J. J,, Pesticide Synthesis Through Rational Approaches

[Washington, DC: ?, 1984).

McFadden, W.H. and Lammert, S. A., “Techniques for Increased Use of Thermospray Liquid Chromatography—Mass Spectrometry, ” J.

Chromatog. 385:201-211, 1987.

Menn, J.J. and Borkovec, A. B,, “Insect Neuropeptides: Potential New Insect Control Agents,”

Jour. All Union Chem. Soc, in press.

Menn, J.J. and Christy, A. L., “New Directions

in Pest Management,” Proceedings US/USSR

Symposium on Fate of Pesticides and ChemicaZs in the Environment, J. Schnoor (cd.) (Iowa

City: Univ. of Iowa, 1988).

Menn, J.J. and Henrick, C. A,, “Rational and Biorational Design of Pesticides, ” Phil. Trans. R.

Soc. Lend. B 295:57-71, 1981.

Menn, J.J. and Henrick, C. A,, “Newer Chemicals for Insect Control,” In: Agricultural Chemicals of the Future, BARC Symposium 8, J.L. Hilton (cd.) (Ottowa: Rowman and Allanheld, 1984),

pp. 247-265.

Morrod, R. S,, “Lead Generation: Designing the

Right Approach,” Phil. Trans. R. Soc. Lend. B

295:35-44, 1981.

Nakagawa, A., Iwamura, H., and Fujita, T.,

“Quantitative Structure Activity Relationship of

Insect Juvenile Hormone Mimetic Compounds,”

/. Med. Chem. 27:1493-1502, 1984.

Nakagawa, Y., Kitahara, K., Nishioka, T., et al,,

“Quantitative Structure-Activity Studies of Benzoylphenylurea Larvicides, ” Pestic. Biochem. &

PhysioZ. 21:309-325, 1984.

National Research Council, Board on Agriculture, Regulating Pesticides in Food: The Delaney

Paradox (Washington, DC: National Academy

Press, 1987).

Pickett, J. A., “Chemical Pest Control—The New
135

Philosophy, ” Chemistry in Britain 24:137-142,

1988.

28. Plummer, E. L., “Biphenylmethylpyrethroids: A

Quantitative Structure Activity Relationship Approach to Pesticide Design,” In: Synthesis through

Rational Approaches, 1984.

29. Shalaby, L. M., “Liquid Chromatography/Mass

Spectrometry of the Thermally Labile Herbicides,

Chlorsulfuron and Sulfometuron Methyl,” Biomed. Mass Spectrom. 12:261-268, 1985.

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Solid-Phase Extraction Aqueous Environmental

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32. Westlake, W.E. and Gunther, F. A., Residue Reviews 18:175-217, 1967.

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Agric. Food Chem, 30:854, 1982.
136

Validation of Analytical Methods for Pesticide

Residues and Confirmation of Results

Henry B.S. Conacher, Food Research Division, Bureau of Chemical Safety, Food Directorate, Health Protection Branch, Health and Welfare Canada

Contents

Page

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ...136

Development of Validated Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..137

Quality Assurance Of Data . . . . . .

References . . . . . . . . . . . . . . . . . . . .

Figure

Figure

l. Stages in Method Development

Tables

Table

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,. 140

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,, 141

Page

and Validation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .137

1.FICP Check Sample Program Outline. . . . . . .

2. Confirmation Techniques and Reporting Limi

Compliance Programs . . . . . . . . . . . . . . . . . . . .

Page

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139

s for HPB Surveillance and

...,,. . . . . . . . . . . . . . . . . . . . . . . . . 140

Decisions by governmental agencies based on

analytical data on pesticide residues in food can

have a significant impact on public health and other

socioeconomic factors. It is therefore essential that

this data be of the highest quality and generated

through the application of validated methods inconjunction with a well-designed quality assurance

(QA) program.

Details are given of the varying degrees of validity achievable for analytical methods. These can

range from validation within a single laboratory up

to the demonstration of satisfactory performance

in a collaborative study conducted and evaluated

according to the guidelines established by international standards-setting organizations such as the

AOAC.

The main problems associated with the development and utilization of collaboratively studied methods in pesticide surveillance and compliance activities relate to the plethora of possible pesticide/

commodity combinations and the daunting task of

devising and conducting collaborative studies of

methods to handle such situations. Some details of

the QA program in effect in the Canadian Health

Protection Branch to ensure the production of valid

analytical data are also presented.

Among a variety of other responsibilities, the

Health Protection Branch, Health and Welfare Canada, is accountable for ensuring the safety of the

Canadian food supply–one aspect of which is the

control of pesticide residues in food. The branch

fulfills this responsibility by (1) establishing maximum residue limits (MRLs) for pesticides (and their

metabolizes) in foods, and (2) establishing monitoring programs to ensure compliance with these MRLs

and to assess the presence of pesticide residues for

which no provision exists in the Canadian Food and

Drug Regulations,

The analytical data generated in these programs

form the basis of decisions regarding compliance

that can have a considerable socioeconomic impact.

It is therefore important that the data be of the high-
137

est quality. To this end, an intensive quality assurance (QA) program is in place for pesticide residue

analysis throughout the branch (12). Similar QA programs are in place in other Federal agencies (Canadian Department of Agriculture, U.S. Department

of Agriculture, and U.S. Food and Drug Administration) with responsibilities for ensuring the safety

of the food supply in North America. The Association of Official Analytical Chemists (AOAC) has also

recently published a handbook in this area (15) that

has drawn extensively on the procedures and practices of the aforementioned and other organizations.

Although there are many critical elements in these

QA programs, there are two that refer to the analytical aspects of the validation process:

1. Development and/or use of appropriate validated methods.

2. Use of appropriate quality control systems to

ensure the production of valid data.

It is particularly important to note that the use

of a validated method, although necessary, is not

in itself sufficient to ensure the production of valid

data. Quality assurance of the measurements on an

ongoing basis is also required.

Development of Validated

Analytical Methods

Validation has been defined (17) as the process

of determining the suitability of methodology for

providing useful analytical data.

There are several steps in the process of developing and of demonstrating the validity of an analytical method. These steps can be conveniently broken down into the three stages outlined in figure 1.

As one proceeds from stage 1 to stage 3, the degree of confidence that one can ascribe to the validity of a particular method increases. Stage 3 represents what is generally accepted (2, 3, 8, 16) to

be the highest degree of method validation, i.e., successful performance in a collaborative study conducted according to the guidelines of recognized

international standards-writing organizations, such

as the AOAC (9).

Figure 1 .—Stages in Method Development

and Validation

Stage 1. Estimation of acceptable performance parameters

within a laboratory.

Stage 2. Demonstration of successful performance in limited

interlaboratory studies.

Stage 3. Demonstration of successful performance in recognized collaborative study.

The main parameters, referred to in stage 1, that

should be taken into account have been identified

in several papers (2, 8) and include accuracy, precision, specificity, limit of detection, limit of determination, linear range, and scope. While these parameters have also been thoroughly discussed in

these publications, it is considered important to

reiterate them here, particularly with reference to

the determination of pesticide residues.

(i) Accuracy–a measure of how closely the determined value (generally expressed as the mean

of several determinations) approximates the true

value of the analyte. This is best supported by the

analysis of standard reference materials; however,

the availability of such materials, especially for pesticides in foods, is generally extremely limited. Normally the recovery of added analyte to “blank”

samples of the commodity in question, over an

appropriate range of concentrations, is taken as an

indication of accuracy. For pesticide compliance

work, the concentration range chosen should certainly bracket the MRL. It should also be recognized

that analyte added to a field sample may behave

differently (typically showing higher recovery) from

field-incurred residues. For analysis at the ppb/ppm

level, recoveries of 70 to 120 percent are generally

considered acceptable.

(ii) Precision–the total interlaboratory precision,

or reproducibility, is the most important aspect of

precision because it is a measure of how much allowance should be made for between-laboratory

variability in interpreting results produced by different laboratories. It is possible, however, to have a

measure of one component of this, the within-laboratory precision, or repeatability, by multiple analyses of samples in the same laboratory over a short

time-period. The reproducibility coefficients of variation (CVS) should fall within the range estimated

by Horwitz et al. (7) with the repeatability components being somewhat lower, generally one-half to

two-thirds of the former. For example, at a pesticide residue level of approximately 1 ppm, the expected reproducibility CV is approximately 16 percent and the repeatability CV, approximately 10

percent. Similar values have been found by Smart

(16) in an examination of UK collaborative studies

on pesticide residues.

(iii) Specificity-the ability of the method to measure only what it is intended to measure. In any

method, it is absolutely essential to run reagent and

field blanks to ensure no interfering compound, or

indeed none of the analyte itself, is present. These

blanks should be run for each commodity examined.

To verify the identity and amount of an analyte, it
138

has been suggested that the ideal approach is to utilize two entirely different analytical methods, based

on different analytical principles (1). However, the

availability as well as the characteristics of such

methods often place a practical limitation on the

application of this suggestion. Thus in the pesticide

area, advantage has been usually taken of the following confirmatory techniques:

a) Mass spectrometric confirmation of identity.

b) Use of different detector, i.e., operating under

different principles such as Coulson vs. Electron Capture.

c) Chromatography using different systems.

d) Chemical reaction followed by analysis.

More detailed descriptions of such techniques can

be found in reviews by Cochrane (5) and by Lawrence

(13).

In the past, these confirmatory techniques have

been generally qualitative in nature and have been

used by the analyst to give reassurance that the validated method was in fact measuring the residue that

it was intended to measure. Since only the qualitative aspects were sought, such techniques were not

required to be subjected to the same rigorous assessment as were the validated methods.

Now, with the availability of the smaller, more

affordable benchtop-type mass spectrometers, the

emphasis is moving toward quantitative mass spectrometric confirmation. This necessitates much

more detailed study of the confirmatory technique.

(iv) Limit of Detection–the lowest concentration

of an analyte that the analytical process can be reliably differentiated from background levels. This has

been defined as the level (background level) measured in the field blank plus 3 standard deviations

(2, 3).

(v) Limit of Quantitation (LOQ)–The lowest concentration of an analyte that can be measured with

a stated degree of confidence. This has been defined

as the level measured in a field blank plus 10 standard deviations; however, it is recommended that this

value be established in the laboratory by repeated

analysis of appropriate samples (spiked or endogenous). In collaborative studies, the LOQ of the

method should be considered as the lowest level successfully analyzed in the study. Collaborative studies

have in fact been used to establish the LOQ (14).

(vi) Linear Range–this is generally taken as the

range over which the procedure has been demonstrated to give a linear response, A reproducible

non-linear response, which is the case with certain

immunological procedures, can also be acceptable.

(vii) Scope–the scope of a method refers to the

number of substrates and the number of analytes

to which the procedure can be successfully applied.

Which of these seven factors is the most important depends on the purpose for which the data will

be used. In the Canadian Food and Drug Regulations, there is a general MRL of 0.1 ppm to cover

pesticides for which MRLs have not been established. Thus, in the Health Protection Branch, in

selecting methodology for surveillance and compliance programs, a major effort is directed toward

the development and validation of methods with

acceptable values for accuracy and precision down

to the 0.05 ppm level.

A higher degree of validation (stage 2) for an analytical method can be obtained by participation in

internal (to the organization) or external check sample programs.

Within the Health Protection branch, certain procedures have evolved over the years to validate the

methodology. These have included the exchange

and analysis of individual (generally violative) samples among branch laboratories, and the distribution and analysis of a variety of check samples. The

latter have usually been distributed in connection

with the emergence of certain contentious issues,

such as the recent ethylene dibromide problem, but

plans are underway to increase the frequency of

check sample distribution during normal monitoring programs, For example, a check sample program underway at present involves the distribution

of two commodities, each containing two different

levels of 1,1-dimethylhydrazine (UDMH) to three

Health Protection Branch laboratories. This study

will serve to validate the methodology recently developed (18) for UDMH.

Undoubtedly the major external check sample

program in which branch laboratories participate

is the Federal Interdepartmental Committee on Pesticides check sample program, details of which have

been given in a recent paper (6). The present program outline is shown in table 1.

As mentioned previously, the highest degree of

validation (stage 3) for an analytical method is the

demonstration of its performance in a successful

collaborative study. Current AOAC guidelines (9)

require the successful analysis of at least five samples in six laboratories. The collaborative study approach not only demonstrates that the method can

be applied successfully in several laboratories but

that it can also withstand an objective, rigorous

peer-review process,

However, a collaborative study generally demonstrates validity for only those commodities and

those analytes included in the study—a fact that

presents a major problem in the area of pesticide

residues because of the large number of pesticide/

commodity combinations possible. This, together
139

Table 1 .-FICP Check Sample Program Outline

Sub- Program Substrates Distribution a Pesticides

Soils Soil

Foods

Water

Fish

Forest Substrates

(Insecticides)

Forest Substrates

(Herbicides)

Wildlife

Feeds

Tallow

Strawberries

Potatoes

Standards

Sediments, Water

Fish, Eels, Cod

Liver Oil

Fish, Soil

Balsam Fir

Needles

Soil

Herring Gull

Lipids and

Homogenates

Grains

3 2,4-D

Picloram

Atrazine

4 Captan

Iprodione

Carbofuran

Chlorophenols

Common OCs

Phenoxy Acids

DDE, Mirex

PCBs

Pirimicarb

Aminocarb

Mexacarbate

Carbaryl

Hexazinone

DDE, Mirex, PCBs

Heptachlor

Epoxide

Chlordane

Oxychlordane

Dieldrin

Trial late

Malathion

Carbathion

Permethrin

Lindane

Chlorpyrifos

aNumber~ of check sample projects conducted in last 5 wars

with the wide range of MRLs, would render the design and conduct of collaborative studies to cover

all possible combinations a most formidable task

that cost alone would surely doom to failure.

An excellent example of these difficulties and the

approach taken to resolve them can be obtained

from consideration of a recent AOAC collaborative

study conducted by Krause (10) of a multiresidue

method for the determination of N-methyl carbamate insecticides and related metabolizes in crops,

and a subsequent publication by the same author

(11).

The collaborative study involved the determination of seven methyl carbamates and two carbamate

metabolizes at two levels in two crops: grapes and

potatoes. This study proved extremely successful

and was adopted Official First Action by AOAC.

Nevertheless the collaborative study had only included two commodities and therefore the method

was only validated for these commodities.

To extend the scope of an Official AOAC Method,

a mini-collaborative study can be required demonstrating that the performance parameters generated

in the main study can be met with the additional

commodities and/or analytes. In Krause’s study, the

method had been initially studied successfully by

four laboratories in an interlaboratory trial on lettuce, in effect a mini-collaborative study, thus permitting the scope to be extended to include lettuce.

The main question relates to what is required to

extend the scope of the official carbamate method

to include other carbamates and other commodities. In his subsequent publication (11), Krause describes recovery values obtained over a 3-year

period in four FDA laboratories for seven parent

carbamates and five carbamate metabolizes added

to 14 crops. These data were obtained as part of

the in-laboratory quality assurance programs. In

many cases, the recoveries obtained were similar

to those obtained in the collaborative study. Whether

this data is sufficient to further extend the scope

of the Official Method depends on its evaluation

by the relevant AOAC committee. In the author’s

opinion, some form of interlaboratory study would

be preferable for this purpose.

Similar situations exist with the other multiresidue screening methods for pesticides.

The stage to which validation should be taken depends to a large extent on the use to which the data

will be put, on the urgency with which the data is
-.

140

required and, indeed, on the operational structure

and philosophy of the organization involved.

In general, regulatory agencies, when faced with

important compliance decisions, wish to have data

of the highest quality. There is therefore a preference for fully collaboratively studied methods (stage

3) or, at a minimum, methods that have been subjected to some form of interlaboratory study (stage

2).

However, if the objective of a survey is simply

to assess if a problem exists, a method in the stage

1 category can readily be used. Even in such cases,

agencies with several field laboratories involved in

generating the data generally undertake limited interlaboratory assessment (2 or 3 check samples)

prior to the survey,

The main drawback to the collaborative process

is the length of time required from initiation of the

study to the stage where the method is given official approval. At present, within AOAC, this takes

a minimum of one year. Thus in situations where

the data is required on an urgent basis, and collaboratively studied methods do not exist, many

agencies resort to the use of methods validated to

a lesser degree.

Quality Assurance of Data

It cannot be stressed enough that the adoption of,

and strict adherence to, a sound quality assurance

program is essential toward the production of valid

analytical data. Within the Health Protection Branch,

an important part of this whole QA program is the

use of appropriate quality control systems in conjunction with validated methods to ensure the production of valid data. The quality control systems,

which include the confirmation of results and the

Table 2.—Confirmation Techniques and Reporting

reporting limits required, vary depending on the

nature of the program. The national surveillance

and compliance programs generally have the highest level of quality control.

The national surveillance program is designed to

determine the state of compliance of selected food

commodities in the marketplace with respect to specific pesticides. The pesticides are divided into high,

medium and low priority groups, and emphasis is

placed first on the high priority group. All laboratories involved must ensure that these pesticides

can be determined by the general screening methodology, or by specific methods, (4) by analyzing

samples spiked with a mixture of pesticides at a minimum frequency of 1 in every 20 samples. Commodities used as the spiked sample are required to

be varied throughout the year, and all high priority

pesticides must be included in the spiking mixtures

at least once per year at, or below, the MRL. If these

recoveries are less than 70 percent or if the limits

of quantitation are greater than one tenth the MRL,

or 0.05 ppm (in the case of the 0.1 ppm MRL), it

is concluded that the particular chemical/commodity

cannot be handled by the methodology and/or the

laboratory in question, and steps are taken to investigate and correct the situation. The medium and

low priority groups of pesticides are included as

time permits.

The confirmation techniques and reporting limits

for the surveillance program, together with the corresponding requirements for the compliance program for comparative purposes, are summarized

in table 2.

Additional confidence in the quality of the data

is obtained through continued (and, it is hoped, successful) participation in the internal and external

check sample programs.

Limits for HPB Surveillance and Compliance Programs

Concentration Confirmation

Program of residue (ppm) Report technique

Surveillance <.01 (or quantitation limit,

whichever is higher) no report required

>=.01 - < 1/2 MRL 1 significant figure 1/10 of specimens by level 1• or

by GC/MS

>/112 MRL - MRL 2 significant figures level 2** ● or GC/MS

> MRL 2 significant figures GC/MS

Compliance < 1/1O MRL no report

> 1/10 MRL (or 0.0 ppm,

whichever is higher 2 significant figures GC/MS

“level 1 = quantitative agreement between 2 columns/2 detectors

“ ● level 2 = level 1 plus an additional column or detector, derivative, or other technique
141

1.

2.

3.

4,

5.

6.

7,

References

ACS AdHoc Subcommittee Dealing with the

Scientific Aspects of Regulatory Measurements,

Chem. Eng. IVews, 44: June 7, 1982.

ACS Committee on Environmental Improvement, AnaZ. Chem. 52:2242, 1980.

ACS Committee on Environmental Improvement, Anal. Chem. 55:2210, 1983.

Analytical Methods for Pesticide Residues in

Foods (Ottawa, Canada: Canadian Government

Publishing Centre, Supply and Services Canada,

KIA 0S9, 1986).

Cochrane, W. P., “Chemical Derivatization in

Pesticide Analysis,” Chemical Derivatization in

Analytical Chemistry, R.W. Frei and J.F. Lawrence

(eds.) (New York: Plenum Press, 1981).

Conacher, H. B.S., J. Assoc. Off. Anal. Chem.

70:941, 1987.

Horwitz, W,, Kamps, L. R., and Boyer, K. W., J.

Assoc. Off. Anal. Chem. 63:1344, 1980.

8. Horwitz, W., J. Assoc. Off. Anal. Chem. 65:525,

1982.

9. Horwitz, W., J, Assoc. off. Anal. Chem. 67:433,

1984.

10, Krause, R, T,, J. Assoc. off. AnaZ. Chem. 68:726,

1985.

11. Krause, R. T., J, Assoc. Off Anal. Chem 68:734,

1985.

12. Laboratory Quality Assurance Manual, Field

Operations Directorate (Ottawa, Ontario, Canada: Health Protection Branch, 1977).

13. Lawrence, J. F., “Confirmatory Tests,” Pesticide

AnaZysis, K.G. Das (cd.) (New York: Marcel Dekker, Inc., 1981).

14. Page, B. D., J. Assoc. off. Anal. Chem. 68:776,

15<

16{

17,

18,

1985.

Quality Assurance Principles for Analytical Laboratories, F.M. Garfield (cd.) (Washington, DC:

Association of Official Analytical Chemists,

Inc., 1984),

Smart, N. A., Analyst 109:781, 1984.

Taylor, J. K., Anal. Chem. 55:600A, 1983.

Health and Welfare Canada, Health Protection

Branch, Laboratory Procedure LPFC-147, 1987.
142

Conventional Pesticide Analytical

Can They Be Improved?

Methods:

James N. Seiber, Environmental Toxicology, University of California, Davis, CA

Contents

Page

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ...142

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....142

Pressure Points In Multiresidue Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ...144

Other Stages Of Pesticide Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...........148

Putting It All Together . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................150

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .. .. .. .. .$ .................151

Who Should Do It? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................151

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...152

Figures

Figure Page

1.

2.

3.

4.

5.

6.

The Evolution of Analytical Methodology for Organic Toxicants in

Environmental Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............143

Tradeoffs in Multiresidue Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

Mills Procedure (PAM) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................145

Acetone Extraction Method (Luke) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ...147

CDFA Multiresidue Screen. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...............149

Hypothetical MR Scheme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................151

Pesticide multiresidue analytical methods have

been continually improved and expanded over the

years. Further improvements are possible based

upon recognition of the limitations of existing methods and their modification with new sample-handling and instrumental techniques. For example, the

use of solid phase extraction (SPE) cartridges in

place of liquid-liquid extraction and, perhaps,

Florisil column fractionation might allow for miniaturization, smaller solvent volumes, extended

breadth of applicability, and greater throughout

when integrated into existing multiresidue schemes.

Wide-bore capillary gas chromatography columns

can eliminate the need for some derivatizations and,

when interfaced with autoinjectors and integrating

data systems, can improve throughput and data

quality. High performance liquid chromatography

can be used for fractionation and also for determination of compounds (including some new classes

of pesticides) that can not be gas chromatographed

without derivatization. Mass-selective detection

(GC/MS), particularly in the selective ion mode, can

improve detection limits and the accuracy of analytical results. These types of potential improvemerits will require coordinated research involving

academic, industrial, and regulatory laboratories,

including new levels of funding for the academic

and regulatory sectors. The importance of academic

involvement can not be overemphasized because

of the need to attract a new cadre of well-trained

young scientists into the residue analytical field.

The field of trace analysis, including pesticide residue analysis, has made tremendous advances in

terms of selectivity and detection limits (figure 1)

(9). In the 1940s and early 1950s, gravimetric and

bioassay techniques were the mainstays in ’’trace”

analysis, extending detection limits to the thenfrontier levels of about 1 ppm. These were timeconsuming methods, lacking in compound selectivity but broad-based in terms of responding to

whole classes of chemicals. Calorimetric and spectrophotometric methods held sway through the

1950s and early 1960s, providing improvements in

both detection limits and specificity. Many of these,
143

Figure I.-The Evolution of Analytical Methodology for Organic Toxicants in —

Environmental Sample s

- -

Trend in cost

>1 Bioassay, gravimetry

0.1

0.0’

0.00

<0.0001

Calorimetry, spectrophotometry

Paper, thin-layer chromatography

Gas and HP liquid chromatography

GC/mass spectrometry>

I I 1 I I

1940 1950 1960 1970 1980

Year (approximate)

1

SOURCE: J N SeIber, lW2hbisol To*mBinAgkutidEtim nments, Genetic Toxicology, R A Fleck mdA Hollaender(eds,)

Plenum, NY, pp 219-234

such as the Sheeter-Hailer method for DDT and

Averill-Norris method for parathion, involved extensive derivatization because they required that a

visibly colored product be formed even when the

parent compound was colorless (as was the usual

case), The inroads of chromatography began roughly

in the 1950s with paper and thin layer chromatography (TLC), and for the first time, chemists were

able to resolve in a given sample several individual

chemicals using a single technique without extensive sample preparation-derivatization. Paper chromatography (PC) and TLC were essentially qualitative techniques, best used to screen samples for the

presence or absence of specific compounds. Clinical chemists interested in drug analysis, natural-products chemists interested in plant secondary products, and pesticide residue chemists quickly adopted

these chromatographic techniques. At present, gas

liquid (GLC) and high performance liquid chromatography (HPLC) techniques have largely supplanted

(but have not completely eliminated) PC and TLC.

These are resolution techniques par excellence,

with the added dimension of quite precise quantitation made possible by very sensitive and often

highly selective detectors. A few of these, such as

the thermionic detector, were developed by pesticide residue chemists while others (electron capture, microcoulometric) were popularized just for

the field of pesticide analysis. The now common

use of mass spectrometry (MS) coupled with GC

provides detection limits to 1 ppb routinely, and it

adds the dimension of near-absolute confirmation

of residue identity when somewhat higher residue

levels are encountered. These achievements in sensitivity and selectivity have been costly, such that

equipping a modern laboratory for a broad spectrum of trace analyses requires considerable

capital—several GCS and LCS plus mass spectrometry capability. Figure 1 shows the interplay, tradeoff, and trends in a very general way for the analytical transition from the 1950s to 1980s. Figure

1 omits the important point that many analyses are

now possible that were not possible in the 1940s

and 1950s, examples being provided by volatile

halogenated organic compounds (VHOC) in drinking water and pesticide multiresidue analysis in

foods.

While these sophisticated methods have revolutionized trace analysis in many respects, the analytical process itself has not changed materially in

that all analyses conform to basic steps, or unit processes, which vary little regardless of the application. These steps include extraction to remove the

analyte from the bulk matrix, cleanup of the extract

to remove potentially interfering coextractives,

modification or derivatization to change the analyte to a more readily determinable form (an optional step), and resolution to separate analyte or

a derivative from other chemicals remaining in the

prepared sample. The elements of concentration,

removing a few micrograms of analyte from several grams or kilograms of sample substrate to a

small volume of solvent, and purification, isolating

one or a few specific compounds from the thou-
144

sands present in the raw sample, run through these

steps. The determination phase includes detection

—obtaining a response related to the structural feature and amount of analyte; measurement—relating

the response to a reference standard of the chemical of interest or a close relative; and confirmation—

assuring that the measured response is indeed due

to the analyte and not an artifact or imposter. This

time-honored strategy takes advantage of physical

and chemical properties unique to the analyte or

analyte class: properties of volubility, polarity, volatility, reactivity, and interaction with electrochemical, optical, ionization, or other detectors. Generally, the more properties built in to the analytical

scheme, the more selective and sensitive the analysis. A corollary is that short-cut methods are often

less selective and sensitive, and thus they place

more demands on the detection and confirmation

instrumentation.

The tradeoffs involved in selecting methods can

also be seen in the following types of analysis:

Qualitative (Screening)–What is present or absent.

vs.

Quantitative–How much is present.

Multiresidue—Capable of measuring many chemicals in a given sample.

vs.

Specific—Tailored to just one or a few chemicals

(e.g., a single parent pesticide plus its major

metabolizes).

Regulatory agencies will require and routinely use

multiresidue pesticide screening techniques because they need to ascertain the presence (or absence) of many chemicals in a given sample of unknown origin. Chemical companies, on the other

hand, need specific and quantitative methods to determine the residue distribution and dissipation of

their own specific pesticide products in a given sample set, in connection with EPA registration requirements or their own need to know.

The dilemma in multiresidue methods may be

stated as follows: The method must cover a broad

waterfront of chemical types and matrices. In so

doing, however, the science of the method, reflected

in the analysts’ prime quality control characteristics—detection limits, precision (reproducibility),

and accuracy—becomes diluted. Very often, costs

(both capital and labor) increase as breadth increases. These tradeoffs, summarized in figure 2,

occur with present-day methodology and thus are

responsible, at least in part, for the compromise nature of existing multiresidue methods. But must it

be this way? Are there approaches yet to be found

that will combine low costs and broad applicability with good science? If so, will they involve modiFigure 2. -Tradeoffs in Multiresidue Methods

SOURCE: James SeIber, University of Cahfornia, Davis, CA, 1998

fying methods that now exist (based largely on GC

and HPLC with selective detection) or instituting

whole new technologies (MS-MS or immunoassay)?

In order to answer these questions it is useful to

look at a few leading multiresidue methods in terms

of their advantages and limitations; in essence, to

find their “pressure points” that are amenable to

incremental (or drastic) improvement.

Pressure Points in Multiresidue

methods

Every analytical method has inherent limitations

that may or may not be amenable to manipulation.

As an example, the PAM (Section 201) Mills procedure (figure 3) represents a “middle of the road”

method optimized to provide quality data on pesticides of intermediate polarity and volatility. It has

at least six points where losses of individual pesticides may occur. For example, paraquat is insoluble in acetonitrile and petroleum ether and is thus

lost in the first step (#l). Aldrin has an unfavorable

petroleum ether/acetonitrile partition coefficient

and is thus partially lost in the discard of the petroleum ether when high fat samples are processed (#2).

Very water-soluble pesticides, such as some organophosphates, maybe lost in the back extraction

from aqueous acetonitrile to petroleum ether (#3).

Very nonpolar and very polar pesticides, which survive to the Florisil cleanup, are further lost because

they elute prior to (#4) or after (#5) the three prime

ethyl ether/petroleum ether fractions. Of course,

some pesticides, including several N-methylcarbamates, degrade on Florisil, adding another limita-
.-

145

Figure 3.- Mills Procedure (PAM)

Sample

Extract with

acetontile (low fat)

or petroleum ether (high fat)

Residue Extract

/

Pet. ether/

acetonitrile

n

partition (high fat only)

*

Insoluble

pesticides

#1 Acetonitrile Pet. ether

1. Add salt water

2. Extract with pet. ether

\

E

Very non-polar

pesticides

Aq. acetonitrile Pet. ether # 2

Florisil

6% 15% Spent

OC’s OP’s ON’s Florisil

Pesticides which

do not GC

# 6

SOURCE: James Seber, University of California, Davis, CA, 1988

tion to the Mills procedure. Finally, success in this

procedure depends on gas chromatography of the

cleaned residues, so that non-volatile materials that

do not elute or degrade upon GC (#6) as well as those

showing poor response to the common selective detector will not be determined.

Thus, in essence, only pesticides within a range

of polarity, volatility, and stability characteristics

will survive the Mills determination. In fact, that

number is about 200 (in fatty foods) or 274 (in nonfatty foods) for pesticides, transformation products,

metabolizes, etc. (5). Some pesticides are not recovered at all, or only partially, because of failure in

one or more of the steps described above. Limitations exist in all multiresidue analytical methods,

so that several pesticides simply “fall through the

cracks” in Federal/State regulatory monitoring programs (l). How can the Mills method be expanded?
146

One approach is to alter or eliminate the limiting

steps. For example, loss in the discarded aqueous

acetonitrile phase (#3) could be minimized by using

a stronger solvent (ethyl acetate or methylene chloride) than petroleum ether in the preceding partition. The tradeoff here could be the appearance of

more interfering material in the final extract, which

might raise detection limits. Alternately, this entire

partition could be eliminated and the acetonitrile

concentrated directly for Florisil cleanup. The

tradeoffs here might be increased detection limits

and lower recovery of some volatile pesticides (v.p.

> 10-3 Torr) because concentrating acetonitrile is

more difficult than concentrating petroleum ether.

Finally, one might substitute a solid phase extraction (SPE) cartridge for the liquid/liquid partition,

providing recovery of a slightly broader range of

compounds and reduced volatility losses—the latter because the SPE elution solvent volume is much

less than that used in liquid/liquid partitioning.

Whether the SPE raises or lowers the detection limit

would need to be determined experimentally.

One could thus critique each step in the Mills procedure, optimize to enhance breadth of applicability while maintaining acceptable detection limits,

and in some cases (such as SPE substitution) perhaps achieve savings in time. Our own experience

with the use of SPEs to isolate metabolizes of organophosphorus pesticides from urine (13) would

tend to support the time-savings notion, particularly

because several samples (up to 10) maybe extracted

simultaneously by SPE. Manufacturers are offering gadgets built upon the SPE concept that facilitate simultaneous extraction and elution as well as

concentration of eluate, providing overall savings

of considerable time and amounts of solvents.

The Florisil cleanup can be attacked in several

ways; miniaturized columns are already in wide use

for relatively clean substrates (many non-fatty foods),

thus reducing solvent volumes, and elution and

evaporation times. Deactivated silica gel can be substituted for Florisil to alleviate the breakdown of

certain compound classes on Florisil, HPLC cleanup

methods could be substituted, allowing for expansion of the range of polarities that could be accommodated, minimizing solvent volumes, and alleviating breakdown. HPLC also has the ability to be

automated, which is difficult with gravity column

chromatography. For example, gel permeation column cleanup has been automated and commercialized for use with fatty foods (PAM). Finally, the

Florisil cleanup step could be eliminated entirely

so that the extract normally entering this cleanup

would go directly to GC determination. The tradeoff

here is increased detection limits (because more

“garbage” enters the GC), decreased GC column life

(same reason), and increased chance for misidentification and misinterpretation (because the Florisil

fractionation works to simplify chromatograms and

help in result interpretation). However, the large

time-saving provided by Florisil elimination, in such

procedures as Luke (PAM Section 232.41, Figure

4), has led to reduced use of it for multiresidue

screening. Note also that the Luke method still provides opportunities for loss of pesticides, but fewer

than in the full Mills procedure. As might be expected, the Luke method is applicable to non-fatty

foods and can not be used with electron-capture or

flame ionization GC detection without prior cleanup.

It should also be apparent that various hybrid techniques can be devised in which the extract prior

to Florisil cleanup is injected on GC to screen for

some chemicals or chemical classes (OPs are most

successfully handled) and then subjected to Florisil

before looking for other classes (OCs, for example)

or for GC/MS confirmation.

As a matter of fact, there are many hybrids and

variations of the PAM version of the Mills (and

Mills-Onley-Gaither) and Luke procedures, most of

which are not published. This is good in that innovations are continually possible, but it introduces

some uncertainty in knowing what is the best procedure for a given analysis and in comparing results from one laboratory to another. An in-depth

study of innovations already in practice might yield

clues that could improve the PAM versions of the

multiresidue methods.

For alternate pesticide multiresidue approaches,

it might be useful to see what types of extraction

and fractionation schemes are used for chemical

pollutants other than pesticides, HPLC-based cleanup methods include the silica column used by Wehner et al. (12) for analyzing pesticides in air, which

has also been applied to water (14) including fog

water (3), The column has a long useful lifetime and

maintains its resolution characteristics. It does require periodic calibration with standards to show

where fraction cuts should be made, but this is a

simple matter of injecting a mixed standard. The

polarity range was successfully extended to include

polar glycosides (11) and derivatized glyphosate in

plant extracts (10) by adding a methyl-butyl ether

(MTBE]-THF gradient after the hexane-MTBE gradient. On the negative side, HPLC cleanup requires

sample concentration to a very small volume ( <0.5

ml) prior to injection on the HPLC, does not tolerate suspended particulate matter, and has a sample throughput of only 1 sample/hr/column, There
147

Figure 4.-Acetone Extraction Method (Luke)

Sample (100 g)

1. Blend w/acetone

2. Fitter

Residue

Pesticides not

extractable with

acetone (aq)

#1

Pesticides which

do not GC

# 3

Pesticides lacking

N, P, S, Cl, Br

Fit&a

Extract with

petroleum ether +

dichloromethane

Pet. ether/MeCl2 Aqueous filtrate

Cone. with

pet. ether \

NPD-TSD

n )

(Florisil optional add-on)

# 4

SOURCE: James Seiber, University of California, Davis, CA, 1988

may be ways of increasing the throughput considerably (very short columns) but these remain to be

proven. Also, the 1 sample/hr/column limitation is

somewhat misleading because 24-hour operation

might be possible if an autosampler were used.

A second HPLC cleanup approach used a cyano

column with hexane to acetone gradient. This procedure was developed by Crowley et al. (3) for separating shale oil extracts, and it was used by our

group (4) to separate mutagenic constituents of

smoke particulate matter. It could probably be used

for some pesticide residue analyses requiring fractionation. Other columns/mobile phases could be

used as well.

It is somewhat interesting to note the diverging

paths taken by pesticide residue chemists who have

concentrated on column chromatography for cleanup and fractionation, and chemists involved with

priority pollutant and drug analyses who have used

acid-base partitioning against organic solvents for

cleanup and fractionation. For priority pollutants

in water, the total scheme uses purging with air or

nitrogen to remove volatile pollutants (benzene,

chlorinated solvents, etc.) and then acid-organic solvent extraction to separate acids (phenols) from

base-neutral chemicals. This scheme would probably not be of general utility for pesticides in foods

because (1) few pesticides are volatile or acidic, (2)

some pesticides are not stable to acid conditions,

and (3) the final base-neutral fraction (which would

contain most common pesticides) might not be

clean enough for low-level GC or HPLC analysis.

The common drug schemes use a more complex

acid-base partitioning system, which serves drug

analyses well because so many of these agents are

bases (alkaloids) or acids (barbiturates, salicylates,
148

etc.). Thus, the cleanup technologies in these

schemes are not applicable to the problem of multiresidue pesticide analyses in foodstuffs, where essentially neutral compounds need to be handled.

Other Stages of Pesticide Analyses

Once a residue-containing extract is provided,

with or without cleanup, the steps of resolution, detection, measurement, and quantitation are performed, occasionally after derivatization. The gas

chromatography equipped with selective detection

based upon the heteroatom (halogen, P,N,S) content of the pesticide analytes is the most common

resolution-detection system employed.

Choices here include the following:

Column type —packed vs. capillary, phase choice

Detector type–Flame photometric (S,P)

Thermionic (N,P)

Hall electrolytic conductivity (Cl,

Br, N, S)

Electron capture (halogens)

Voluminous literature exists on the applicability

of each combination. Suffice it to say, the packed

vs. capillary issue is still debated, with more converts to capillary following the introduction of reproducible splitters and megabore columns. These

columns provide greater resolution and greater efficiency than packed columns, both of which generally lower detection limits and increase confidence

in the results. They also minimize breakdown and

irreversible adsorption of the more thermal-labile

and polar pesticides, thus increasing breadth of applicability. They do not, however, have the capacity to accept very dirty extracts that might be chromatographable on packed columns. This represents

another tradeoff, although in spite of it the technology is clearly leaning toward more capillary and

less packed column use.

Regarding column phases, analysts already have

a large selection (summarized in PAM Section 301),

with the only new developments occurring in adapting conventional phases (or mimics) to fused-silica

capillary columns.

Summarizing for columns, high load, bondedphase megabore columns will suffice for virtually

all GC systems and, through improvements in technology that are rapidly emerging, will extend the

applicability of GC to even broader ranges of pesticide types, They will also minimize the need for

derivatization (a time-consuming and error-prone

procedure best avoided if possible) of some phenolic, carbamate, and polar metabolize chemical classes,

More work in proving these points will pay rich

dividends in improving conventional methods.

In GC detection, many improvements in virtually

all detectors have occurred in the past 5 years, and

these are being rapidly adopted by residue chemists.

They include the following:

1.

2.

3.

FPD detection limits have been improved almost

tenfold. This is still the most reliable system for

OP and S-containing pesticides,

Hall-type electrolytic conductivity detectors

have improved dramatically and are now clearly

the first choice for organohalogen compounds

and near first choice for organonitrogen compounds.

The thermionic NP-TSD shows steady improvements and represents a viable choice for OP

and ON analyses.

4. The pulsed-mode Ni63 EC is a vast improvement over earlier EC detectors and is still useful for some organohalogen compounds, particularly the more volatile ones resolved by

capillary GC.

Newer detectors that may supplement the above

improvements include the following:

1.

2.

The

cial

Photoionization, particularly for aromatic compounds lacking heteroatans and for some polysulfur and polyhalogen compounds. There is

now more than one supplier of this promising

detector,

Mass selective (MS) detectors, for virtually all

compounds,

mass selective detector, or MS, is worth spenote because of its universality, confirmatory

power, rapidly improving detectability (particularly

in the selective ion or SIM mode), and a healthy

trend to lower priced, user-friendly systems of increasing ruggedness.

Many analysts have shied away from MS, including the mass selective detector (MSD) Ion-Trap version, and other GC/MS systems, feeling that it is

more suited to dedicated analyses for a single analyte or small analyte clusters than for the range of

analytes potentially present in a multiresidue sample of unknown origin. This is becoming a less valid

objection because new MSs can be programmed to

shift rapidly between pre-selected masses as the

chromatogram develops, thus covering the broad

range needed for many applications. Some industrial and contract labs have moved more to MS, to

the point of replacing element-selective detectors.

This is a very healthy trend and should be encouraged by increasing research funding in the area of

tailoring multiresidue schemes to be compatible

with the MS.

Aside from much improved versions of traditional

pesticide analytical methods in the areas of capillary columns, improved selective detectors, and the
149

GC mass spectrometry-based systems, other conventional techniques and a few relatively new approaches are finding increasing applications in pesticide multiresidue methods. Chief of these is HPLC,

which has become close to routine in handling analysis of some pesticides that are not amenable to GC,

or for which HPLC provides an alternative to derivatization for GC. For example, some N-methylcarbamate insecticides (and their metabolizes), for which

derivatization and GC represented the only viable

approach just a few years ago (8), are screened by

HPLC using either direct UV/fluorescence detection, or detection following automated postcolumn

derivatization. The multiresidue procedure of the

California Department of Food and Agriculture (2)

(figure 5), for example, integrates the use of SPE

isolation with postcolumn derivatization HPLC,

with detection limits for eight carbamates in the

range of 0.2-0.5 ppm. Moye (6) provided other examples, including for glyphosate, phenoxy acids,

and substituted ureas. Once again, technical improvements in HPLC columns and detectors have

provided increased resolution, detectability, and

reliability. Capillary column HPLC and supercritical fluid chromatography promise further advance:

capillary columns in extending resolution and detectability further, and SFC in the ability to interface with the selective GC detectors (an area where

HPLC is normally at a disadvantage relative to GLC).

HPLC-MS is also improving, particularly with thermospray and other new interfacing systems, but is

not yet competitive with GLC-MS in detectability

and confirmatory power.

Still other instrumental advances may find future

use in multiresidue analysis, including the following:

● Headspace GC—Volatile pesticides (methyl bromide, ethylene dibromide, phosphine)

● GC-Fourier Transform 1nfrared—SemivolatileFigure 5. -CDFA Multiresidue Screen

Sample (high moisture)

1. Blend with acetonitrile

2. Fitter

1

Residue

/

Acetonitrile

I

separate

aliquots

Filtrate

Add NaCl,

I

separate phases

Aqueous NaCl

\

1. Exchange to benzene Exchange 1. Exchange to methanol

2. Add Attaclay, settle to acetone 2. Pass thru C-18 SPE

EC-GLC NP or FPD HPLC/post-column

for OC’s GLC for OP’s derivatization for

carbamates

SOURCE: Jamas SaIbar, Univerwly of California, Davis, CA, 19S8
150

volatile pesticides; can be interfaced with GCMS (GC-IR-MS)

● Multidimensional GC—More rapid screening

for varieties of pesticides in single extracts.

● Tandem mass spectrometry (MS-MS)—Screening samples for classes of chemicals; confirmation.

● High resolution mass spectrometry (HRMS)—

Screening samples for classes of chemicals;

ultra-low level detection of specific chemicals;

confirmation.

. Immunoassay—screening samples for selected

classes of chemicals, particularly those not

amenable to low-level GC or HPLC analysis.

Finally, a few comments should be made on automation, and other time- and labor-saving approaches.

Autoinjectors for GC and autosamplers for LC are

commercially available in much improved versions

over early devices introduced in the 1970s. Both can

be considered routine and have extended sample

throughput to 24-hour operations. A necessary adjunct to autoinjectors is a programmable integrating data system for data compilation, eliminating

the need to have all peaks on-scale for quantitation.

Another convenient adjunct is the use of internal

standards (nonpesticide surrogates that chromatography similarly to pesticides), bypassing the need

for extensive standard curve preparation and reinjection of “out of range” samples.

Thus, microprocessor-controlled GLCs and HPLCs

with autoinjectors and computer data systems are

seen in increasing frequency in pesticide analytical laboratories and, combined with internal standards, can make large improvements in the time and

costs of residue analysis. This trend will continue,

as “smart” systems that identify (based on retention time) and quantitate suspected residues with

less operator involvement are further refined and

utilized. The data systems of GLC and HPLC instruments have the added advantage of providing records, which can help fulfill good laboratory practices requirements.

The use of SPE cartridges was mentioned previously as a sometimes more rapid and generally

solvent-saving device in the extraction-cleanup

phases of analysis. SPEs also provide an opportunity to conduct some sample preparation in the

field. For example, we extracted water samples for

pesticide residues by pumping water through C-18

SPEs immediately after the samples were taken in

the field; very little extra time was added to that

required for sampling and only the small SPEs

needed to be transported to the lab for completion

of analysis by GLC. This approach could perhaps

be extended directly to milk, juices, and other fluid

foodstuffs, and perhaps even to solid foods if a solvent extraction operation were set up close to the

point of sampling.

Improved gadgetry for solvent concentration is

gradually replacing the very clumsy, labor-intensive

rotary evaporators and Kudera-Danish-Snyder column steam concentrators. For example, the N-Evap

proved useful in simultaneously concentrating

many samples of small solvent volumes when first

introduced in the 1970s. A recently introduced

programmable sand bath evaporator should also

find use, particularly for concentrating aqueous

samples. In fact, any move by technology toward

smaller sample sizes and extract volumes should

save time and decrease chances for in-house contamination because of the smaller glassware requirements.

Taking what appears to be the best of existing MR

schemes, and extending to sample miniaturization

and SPE cleanup-fractionation, leads to the hypothetical “method” in figure 6. The hypothetical

method is broad-based in terms of handling pesticides over a range of volatilities and polarities, and

it is quick because of lower volumes handled and

the elimination of liquid/liquid extraction and solvent evaporation.

In this approach, all GCs are equipped with megabore capillary columns, autoinjectors, and data systems. LCs are equipped with short, small particle

columns, autosamplers, and UV and fluorescence

detectors with and without postcolumn derivatization. The GC/MS is programmed in the selective ion

mode to search for ions diagnostic for individual

pesticides. Quantitation is done vs. internal surrogate standards added to the first extract. Recoveries are calculated for the internal standards by

occasional external standardization. Fluid samples,

high fat, and low fat samples could potentially be

accommodated with some modification of the first

extraction step. All steps would need research and

developmental optimization, using a variety of substrates and pesticide types. The point here is that

most pesticide residue chemists could come up with

a scheme that, conceptually, improves on existing

methods by instituting newer technologies and miniaturization. Whether these conceptual schemes

could extend multiresidue methodologies to new

levels in the parameters in figure 1 is a question

worth asking—and perhaps worth investing of public funds to answer.
151

Figure 6.- Hypothetical MR Scheme

Sample (1-10 g)

I

1. Blend with acetonitrile

2. Fitter

Headspace Extract

GC Aliquot (save)

(volatiles)

/

Not retained

/

HPLC

(acids, polars)

\ – r

1. Concentrate

2. Add water

oSPE

Elute With

\

high polarity

I

Elute solvent

with low

polarity Extract

solvent

\

Extract \

HPLC

(carbamates)

+

GC-Hall (OC)

GC-FPD (OP, S)

GC-NP-TSD (OP, ON)

or

GC-MC (above, plus

pyrethroids, biphenyl, etc)

SOURCE: James S@ber, Umversity of Cahfcma, Davis, CA, 19SS

Conclusions

Can existing analytical methods be improved?

The answer is certainly yes, and in fact they are

continually undergoing improvement as new GC,

HPLC, and MS systems are introduced and as new

chemicals are included in the existing schemes.

How can they be improved? The following are

offered as potential elements to improvement:

1.

2.

Miniaturization—Offers savings in time by

processing smaller volumes, increases the possibility of automation, and might extend breadth

(yet to be proved) through the use of commercial cleanup columns (HPLC or SPE cartridges).

GC Technology—Wide bore capillary columns

interfaced with element-selective detectors will

minimize the need for derivatization and pro3,

4.

vide more efficient resolution, thus lowering

detection limits. When used with auto-injectors

and integrating data systems, throughput and

data quality will improve.

HPLC Technology—Will focus on those compounds that cannot be determined by GLC; critical need for selective HPLC detectors that go

beyond UV, fluorescence, and electrochemical

(SCF improvements will help here); if detectors

were available, promises major improvement

on breadth of MR technology. Even without

super-selective detectors, HPLC will find specialty use in removing problem interferences

and in postcolumn derivatization for specific

classes of chemicals.

MSD Technology—This represents a real opportunity because this is an affordable, hereand-now technology that only needs a few welldesigned studies to show applicability, particularly in the SIM mode with programmed ramping of ion masses through the chromatogram.

MSD can be used as a detector (SIM mode) and

also for confirmation (Scan mode).

Who Should Do It?

Industry has a responsibility for fitting new compounds into multiresidue schemes, but not for the

development of the schemes themselves. Federal

labs should take the lead, set the goals, and conduct

the validation (with AOAC or other assistance). But

they should not shoulder the burden of discovery

and development alone.

A well-conceived, extramural funding program

is needed, allowing for participation by academic

institutions, research institutes, and state lead agencies. New funds in the range of $10 million would

be needed, with half devoted to upgrading the equipment and scientific expertise in the residue labs of

state agencies and the four regional Leader Laboratories of the USDA- CSRS - State Experiment Station’s Minor Use Registration Program (IR-4), and

half to competitive funding on a Request for Proposal (RFP) basis. Categories for the RFP would be

the four mentioned above, in this section, and also

a fifth dealing with extending multiresidue methods to new classes of pesticides (sulfonylureas,

pyrethroids, etc.). Academic involvement is crucial,

both to supply new ideas and also to stimulate involvement of graduate students and post-doctorals

who will provide the invigoration needed for a

longer-term, sustaining program of residue analytical excellence, which is needed over the long-haul.
152

10

2.

3.

3.

4.

5.

6.

7.

References

Ambrus, A. and Thier, H. P., “Application of

Multiresidue Procedures in pesticides Residues

Analysis,” Pure and Applied Chem. 58:1035-

1062, 1986.

California Department of Food and Agriculture,

Multiresidue pesticide Screens, California Department of Food and Agriculture, Sacramento,

CA, 1988.

Crowley, R. T., Siggia, S. and Uden, P. C., “Class

Separation and Characterization of Shale Oil by

Liquid Chromatography and Capillary Column

Gas Chromatography,” Anal. Chem. 52:1224-

1228, 1980.

Glotfelty, D. E., Seiber, J.N. and Liljedahl, L. A.,

“Pesticides in Fog,” IVature 325:602-605, 1987.

Mast, T. J., Hsieh, D.P. and Seiber, J. N., “Mutagenicity and Chemical Characterization of Organic

Constituents in Rice Straw Smoke Particulate

Matter,” Environ. Sci. Technol. 18:338-343,

1984.

McMahon, B.M. and Burke, J. A., “Expanding

and Tracking the Capabilities of Pesticide Multiresidue Methodology Used in the Food and

Drug Administrations Pesticide Monitoring

Program,” j. Assoc. Offic. Anal. Chem. 70:1072-

1081, 1987.

Moye, H. A., “High Performance Liquid Chromatographic analysis of Pesticide Residues, ”

Analysis of Pesticide Residues, H.A. Moye (cd.)

(New York: Wiley-Interscience, 1981), pp. 333-378.

Pesticide Analytical Manual, Department of

Health and Human Services, Food and Drug Administration, Washington, DC, Sections 201.01,

8. Seiber, J. N., “Carbamate Insecticide Residue

Analysis by Gas-Liquid Chromatography,” Analysis of Pesticide Residues, H.A. Moye (cd,) (New

York: Wiley-Interscience, 1981), pp. 333-378.

9. Seiber, J. N., “Analysis of Toxicants in Agricultural Environments,” Genetic Toxicology, R.A.

Fleck and A, Hollaender (eds.) (New York: Plenum, 1982), pp. 219-234.

10. Seiber, J. N., McChesney, M. M., Ken, R., et al.,

“Analysis of Glyphosate Residues in Kiwifruit

and Asparagus Using HPLC of Derivatized

Glyphosate As a Cleanup Step,” J. Agric. Food

Chem. 32:678-681, 1984.

11, Seiber, J, N., Brewer, L. P., Lee, S. M., et al.,

12

“Cardenolide Connection Between Overwintering Monarch Butterflies from Mexico and Their

Larval Food Plant,”J. Chem. EcoZ, 12:1157-1170,

1986.

Wehner, T., Woodrow, J. E., Kim, Y-H., et al.,

“Multiresidue Analysis of Trace Organic Pesticides in Air,” Identification and Analysis of Organic Po]Zutants in Air, L.H. Keith (cd.) (Boston,

MA: Butterworth, 1984), pp. 273-290.

13. Weisskopf, C. and Seiber, J. N., “New Approaches

14

to the Analysis of Organophosphate_s in the

Urine of Field Workers,” Paper presented at the

194th National Meeting, American Chemical

Society (AGRO 141), New Orleans, LA, Sept. 3,

1987.

Woodrow, J. E., Majewski, M. S., and Seiber, J. N.,

“Accumulative Sampling of Trace Pesticides

and Other Organics in Surface Water Using

XAD-4 Resin,” J. Environ. Sci. Health 21:143-

164, 1986,

and 232.41.
153

Techniques for and the Role of Screening

Pesticide Residue Analysis

Richard L. Ellis, Director, Chemistry Division, Food

Department of Agriculture, Washington, DC

Contents

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Screening Method Concepts . . . . . . . . . . . . . . . . . . . .

Attributes of Screening Methods . . . . . . . . . . . . . . . .

Quality Assurance . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Detection Systems for Screening Methods . . . . . . . .

Safety and Inspection Service, U.S.

Page

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 153

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

. . . . . . . . . . . . . . . . . . . . . . . . . . . . 155

,..,.. ,..,,.0 . . . . . . . . . . . . . . . 156

. . . . . . ,.,.... . . . . . . . . . . . . . . , 156

Integrating Screening Methods into Multiresidue Regulatory Programs . . . . .......159

Constraints on Use of Rapid Test Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ...160

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...160

Figure

Page

1. Decision Tree for Regulatory Control Program. . . . . . . . . . . . . . . . . . . . . . . .......161

Abstract

Analytical testing for residues has progressed

from relatively insensitive bioassays to highly technical procedures combining attributes of computer

science, electronics, materials sciences, and biotechnology. Consumers, consumer action groups,

the National Academy of Sciences, Congress, and

others have highlighted public health concerns

about the detrimental effects of pesticides residues

in food, water, and other parts of our environment.

Regulatory programs are designing improved statistical-based sampling programs to ensure a safe

and wholesome food supply. These programs highIight the magnitude of testing required to accomplish that goal. Testing procedures, once the domain

of relatively complex quantitative and confirmatory

techniques run in well-equipped laboratories with

trained personnel, are not adequate to meet analytical demands with available resources. Rapid test

systems employing advanced technology are being

produced that can make residue testing programs

responsive to this need. Regulatory statutes require

a preponderance of information to support enforcement responsibilities. Thus, integrating rapid test

procedures with quantitative and confirmatory

methods is needed. Rapid testing procedures may

be used by personnel nonexperienced in analytical

sciences. This requires strong quality-assurance and

quality-control programs to ensure proper design

and use of rapid test methods. Regulatory-agency

policy development on roles for these screening

methods as well as other technological developments for testing procedures will influence development and application for improved public health

analytical testing programs now and in the future.

Background

Modern agricultural production uses commercially available pesticides to combat a variety of

weeds, insects, fungi, and other agricultural pests.

These pesticides contribute substantially to the high

level of agricultural production we currently enjoy.

As a result, consumers are exposed to pesticides,

usually in minute quantities, in several food groups

including meat, dairy products, fruits, vegetables,

dried food goods, most processed foods and many

other household staples, Some pesticides, however,

are considered as either acutely or chronically toxic

to humans and other segments of the environment,

and they pose potentially serious health risks to nontarget organisms and species. This presents a significant regulatory responsibility to public healthrelated agencies as well as to Congress.

The magnitude and complexity of the regulatory

responsibility is well developed by the Congressional Research Service 1986 report and the National Academy of Sciences report, “Regulating Pes-
154

ticides in Foods.” Regardless of the number of

pesticides registered for use on agricultural products, the number is small compared to the more than

8,000 food tolerances listed in the Food, Drug and

Cosmetic Act, Section 408 and 409 (2). From data

supplied by EPA, 53 of these pesticides have active

ingredients identified as oncogenic or potentially

oncogenic (2). This does not include some of the

chlorinated hydrocarbons considered as oncogenic

in animals or humans. Further, FDA has classified

81 compounds through its surveillance index as

warranting residue monitoring because of their potential health hazard. Based on the GAO Domestic

Food Report (3), 30 of these received little or no residue testing between 1979 and 1985, and several pesticides included in the two highest classes as potential health hazards are not covered by the five

current FDA multiresidue methods, although these

residues may be analyzed by other methods or other

programs. Recognizing the universe of pesticides

used on foods, there is agreement that more residue methods are needed for monitoring purposes.

This becomes more important as higher levels of

sampling are called for to improve confidence that

regulatory agencies are providing greater assurance

of a safe food and water supply to consumers.

Screening Method Concepts

Analytical methods play an important role in food

production inspection systems to protect public

health. A universal analysis scheme that can simultaneously quantify the presence of all compounds

or classes of compounds of interest in foods, animal tissue, or fluid with acceptable accuracy and

correctly identify the analyte or analytes would be

a desirable, unified methods approach for regulatory control agencies. Yet at present, there are very

few analytical procedures available to regulatory

agencies that simultaneously quantitate and confirm the identity of such residues. Until universal

methods are available, regulatory programs will

have to employ methods with individual attributes

of presumptive presence, quantitation, and positive

identification. To accomplish this goal, methods

with different attributes must perform in concert

for a highly effective residue program regardless

of individual regulatory mandates.

Terms such as confirmatory, reference, quantitative, semiquantitative, screening, rapid, and presumptive methods are well known. An alternative

to the potential difficulty of categorizing methods,

and the stigma associated with these descriptive

terms, is to define the methods independent of intended purpose, according to the attribute or qualities of method performance. Attributes and qualities of three levels of analytical methods are relevant

to support regulatory programs. Though the focus

will be on screening methods, a brief description

of the method types is needed to understand their

interrelationship.

Level I methods incorporate the ability to quantify the amount of specific analyte or class of analytes and positively identify their presence in a single analytical procedure. These are assays with the

highest level of credibility and are unequivocal at

the level of interest. They maybe single procedures

that determine both the concentration and identity

of the analyte, or combinations of methods for determining and confirming a residue for definitive

identification, These methods are most commonly

identified as confirmatory methods.

Level II methods are those that are not unequivocal but are used to determine the concentration of

an analyte at the level of interest and to provide

some structural information. For example, these

methods may employ structure, functional group,

or immunochemical properties as the basis for the

analytical scheme. These methods are often reliable enough to be used as reference methods. Level

II methods provide a quite acceptable approach for

residue testing.

They may be used to corroborate the presence of

a compound or class of compounds. Thus, two Level

II methods may provide information suitable for

Level I attributes, providing they employ different

chemical technologies. The majority of analytical

methods now available and used by regulatory control agencies are Level II methods, These methods

are usually the quantitative analytical methods used

in laboratories for regulatory control programs.

Level III methods are those that generate imperfect, though useful, information. These testing procedures detect the presence or absence of a compound or class of compounds at some designated

level of interest and often are based on non-instrumental techniques for analytical determination. Results on a given sample are not as reliable as Level

I or II methods without corroborating information.

Level III methods may, for example, provide reasonably good quantitative information but poor

compound or class specificity or identity, or they

may provide strong or unequivocal identification

with very little quantitative information. Level III

methods are not poorly described or sloppy methods; rather, they must have defined operating characteristics of reliable performance. Many microbiological assay procedures and immunoassay test
155

systems may fall into this category. They are used

because of convenience and potential suitability to

non-laboratory environments, analytical speed,

sample efficiency through batch analysis, portability to non-laboratory environments, sensitivity, and

the ability to detect classes of compounds. The hallmark of Level III-type methods is that action based

on individual positive results require substantiation

using Level I or II methods as required by the uncertainty of an individual result. However, epidemiological information may provide substantive

data, reducing the uncertainty of individual results.

These are typically screening or rapid test methods and may offer several advantages to a regulatory control program.

The reliability of Level III methods should be

measured in part by their performance characteristics as well as their ability to handle relatively large

numbers of samples within a given timeframe. Two

key characteristics requiring definition include

their percent of false positives (reporting a positive

response when no analyte is present) and percent

of false negatives (reporting a negative response

when the analyte is present) when measured against

a validated quantitative assay in a statistically designed protocol to derive the test method operating characteristics. When the operating characteristics are defined for false negative and false positive

results, the operating range of the screening method

may be established. Individual programs may select those false negative and false positive values

to suit their particular program needs. The percent

of false negatives must be quite low at the levels

of interest (less than 5 percent), while slightly more

flexibility may be acceptable for false positives for

screening tests. A minimum level of residue detection can be described based on a balance between

these two parameters.

Attributes of Screening Methods

Methods suitable for regulatory purposes must

be reliable. To ensure analytical reliability, performance characteristics of a method must be determined by multilaboratory evaluation. In most cases,

minimum standards should be set, designed to fit

the needs of specific program requirements. By consensus with public health standard-setting organizations or agencies, the principal attributes of analytical methods are specificity, precision, systematic

error, and sensitivity. Other attributes relevant to

screening methods will be described as well.

Specificity is the ability of a method to respond

only to the substance being measured. A residue

control method must provide for unambiguous

identification of the compound being measured.

One set of measurements of specificity is the percent of false positives and false negatives. This characteristic is often a function of the measuring principle used and the analyte functionality—key factors

for rapid test methods. Methods should be able to

qualitatively differentiate the analyte from analogues or metabolic products of the compound(s)

of interest under the experimental conditions employed.

Precision is a measure of the variability of results

when the method is applied to separate portions of

a homogeneous sample. Precision is usually expressed as standard deviation. This term is sometimes used to describe other method characteristics such as limit of detection, limit of decision (4),

and limit of reliable measurement (5). Another useful term is the relative standard deviation because

it is relatively constant over a considerable concentration range (an order of magnitude, for example),

ideally covering the level of interest.

Systematic error is analytical method bias, the

difference of the measured value from the true, assigned, or accepted value (mean 8 value). It is commonly expressed as the percent recovery of added

analyte to a sample blank. At relatively high concentrations, recoveries are expected to approach

100 percent. At lower concentrations and particularly with methods involving a number of steps,

recoveries may be lower. Regardless of what average recoveries are observed, low variability is a

desirable feature. Commercial rapid test systems

should be designed so that parallel curves for standard solutions of the analyte and sample extracts of

analyte added to a sample are routinely achieved.

Accuracy refers to the closeness of agreement between the true value and the mean result. The accuracy requirements of different types of methods

will vary with the use being made of the results.

For screening methods, characteristics of false positive results and false negatives define a methods

operating range.

The sensitivity of a method is a measure of the

ability to discriminate between small differences in

analyte concentration. A common practice is to define sensitivity as the slope of the calibration curve

with known standards at the level of interest.

Beyond these method characteristics are a number of collateral criteria particularly suitable for

screening methods for regulatory control programs.

Methods should be rugged or robust, cost-effective,

relatively uncomplicated, portable and capable of

handling a set of samples simultaneously in a timeeffective manner. Ruggedness of a method refers
156

to its capability to be relatively unaffected by small

deviations from the established values in the use

of reagents, quantities of reagents used, time factors for extractions, and reaction or temperature.

This does not, however, provide latitude for carelessness or haphazard techniques. Cost-effectiveness refers to use of relatively common reagents,

efficient use of resources, and using instrumentation commonly used for trace environmental analyses. A method of being relatively uncomplicated

refers to use of simple, straightforward mechanical or operational procedures throughout the

method. Portability is the characteristic of the

method that enables it to be transferred from one

location to another without loss of established performance characteristics. The capability to analyze

a set of samples simultaneously reduces the analytical time requirements of sample analysis. This

is particularly important for screening methods in

which large numbers of samples are to be analyzed

in short or fixed timeframes.

The importance of establishing the attributes and

performance criteria cannot be overemphasized. It

provides the necessary information to allow regulatory control officials to develop and manage programs responsive to their public health responsibilities. Performance criteria for analytical methods

also provide a basis for good management decisions

in future planning, evaluation, and product disposition.

Quality Assurance

Regulatory control agencies responsible for monitoring foods are routinely made aware that any

analytical discrepancy may require the inevitable

defense of our analytical programs. A principal objective becomes one of assuring we have a well

planned and executed quality-assurance program.

Quality assurance is an important part of all regulatory control programs. With screening methods,

or any rapid test system, established policies and

procedures are needed to ensure that these methods are being properly conducted and the testor is

evaluating the test response in the appropriate manner. The implications of poor performance of rapid

test methods would be difficult for regulatory control agencies to deal with.

Quality assurance begins with the method development process. Activities include experimental optimization of each procedural step or manipulation

to determine the critical control steps—those having a substantial impact on method performance.

Other activities include identifying when an analytical method may be stopped without adversely

affecting the results; determining the ruggedness

or process variability that maybe employed in any

particular method step without reducing the method’s performance; and determining the sample requirements necessary to ensure reliable, interference-free results. Instrument parameters should be

optimized and a mechanism to test instrument performance established if instruments are required.

Mass transfers in the procedure should be minimized, Lastly, the method must be written in thorough, concise, unambiguous language. These factors will facilitate method transfer and training for

end-users of a method into a regulatory program.

The focus on quality assurance cannot be overemphasized. In the long-term, it is less expensive to

do it right the first time. It ensures credibility to

a regulatory program and esprit de corps among

analysts.

Detection Systems for Screening

Methods

Two important reasons for using screening methods are 1) their capability to analyze a relatively

large number of samples in a given unit of time,

and 2) their robust nature. This latter characteristic allows latitude for using screening methods in

non-laboratory surroundings. In these instances,

methods will often be used by individuals not necessarily experienced in analytical chemistry techniques. This places a constraint on certain types of

methodology. It limits use of certain types of equipment, instruments, and reagents. Further, methods

need simple, unambiguous test instructions that will

enable a testor to correctly prepare the test material, conduct the analysis, and interpret and report

the test findings. Process controls defining critical

steps in the test procedure are very relevant to the

success of such a testing program.

Thin layer chromatographic procedures satisfy

a significant number of desired attributes for screening methods. The capability to analyze a set of samples in a given timeframe is usually higher than

other common chromatographic systems. There is

a-wide variety of absorbents, chromatographic solvents, and reagents facilitating residue detection.

In addition, residue detection is a static process

rather than a dynamic one; quality assurance is easier because control samples and reference standards can be analyzed simultaneously with the test

samples. A comprehensive review on thin layer

chromatographic systems and procedures has been

published recently (6). It describes an extensive array of systems for pesticide analysis. One that has
157

been reduced to practice for a regulatory program

consists of thin layer chromatography for separation of 12 organophosphate pesticides using cholinesterase enzyme inhibition for residue identification (7). A recent project with Food Safety and

Inspection Service (FSIS) for screening chlorinated

hydrocarbon pesticides employing thin layer chromatography with a variety of detection systems for

chlorine (including many in reference 6) was not

successful because the sensitivity at the level of interest was not attainable (8). A further complication

was the sample extraction procedure from animal

fat being too complicated for use in a nonlaboratory situation. Detection systems focused on the

chlorine atom because of the relatively high chlorine content in the compounds of interest. A successful application of thin layer chromatography

for a rapid field test has been developed for sulfamethazine by FSIS. Although these are limited applications for regulatory programs, this technique

offers promise for the future as new reagents improve sensitivity and thin layer chromatography

systems provide new approaches for effective sample purification and analysis.

The detection limits of most color-producing or

fluorescent-generating reagents provide sensitivity

at low microgram per gram (μg/g, ppm) concentrations. Reagents using enzyme inhibition allow detection limits in some systems at low picogram per

gram (pg/g, ppb) concentrations. For example, many

herbicides employing photosynthesis inhibition as

a mechanism of action have been detected at picogram (10-12g) levels using plant chloroplasts and

a reduction-oxidation chemical indicator. In corn,

potatoes, and carrots, detection limits without

purification of the sample extract were less than

10 pg/g (ppb) (9). Classes of herbicides adaptable to

this detection system include triazines, phenylureas,

phenylcarbamates, 13 uracils, and acyl anilides.

This suggests the possibility of broad-spectrum

screening tests suitable to nonlaboratory use.

Immunobased assays are emerging as promising

screening test methods. Test systems for a wide variety of organic residues in soil, water, food, plant,

and animal tissues are being developed by a number of companies in the United States. These tests

are being developed in rapid, very sensitive, easy

to use, and usually highly specific formats. They

show promise for rapid onsite testing as qualitative

assays while some are now being designed for fast,

quantitative laboratory tests. Their designed specificity, which is commonly very high, generally

allows use of relatively crude samples as a test material and makes them attractive for use in nonlaboratory environments. Generally, the cost of

these assays is lower than traditional analytical laboratory methods. However, they are still dependent

on sample preparation. Nevertheless, potential persample cost for such assays is less than $15,00, including administrative costs. Instrumental methods

are usually $50.00 or more for similar analyses. The

major constraint of these assay systems is their relatively high cost of development. It is estimated that

they become practical economic investments by

economy of scale, when 100,000 tests per year are

run (10).

Within our current regulatory and statutory environment it is not reasonable to expect registrants

of pesticides or other chemical entities used in food

production to voluntarily provide screening methods. There is little interest in developing multiresidue methods, in particular, that may be capable of either identifying or quantitating residues in

food products that may include a competitor’s product. Where a residue control problem exists or is

likely to exist, Federal agencies commonly take the

initiative for developing these methods. Because of

the costs involved, prudent decisionmaking on priorities is essential. It must be understood that in

certain instances, other metabolism or metabolic

research may be needed to provide a basis for developing an analytical system responsive to regulatory control needs.

Opportunities do exist to stimulate methods development in the private sector. Examples include

the recent legislation allowing commercialization

of Federal government supported patents, federally

supported research contracts and grants, and advertisement for commercially available analytical

technologies. Within the Food Safety and Inspection Service, the last two have been extensively explored with measurable success. It is likely other

Federal agencies have similar and possibly other

opportunities to stimulate private sector interest.

A known long-standing or highly publicized residue problem often generates heightened interest.

The big advantage of rapid test systems is their

simplicity allowing tests to be performed by testers

that are not highly experienced in diagnostic or analytical procedures. A disadvantage on occasion is

that they are designed specifically for only one compound and require separate test systems for a class

of pesticides. In some instances, sufficient crossreactivity to a class of pesticides will allow other

compounds to be detected, usually at higher concentrations in a sample matrix. Thus, there is some

tradeoff for development by laboratories and use

in regulatory programs.
158

It is often possible to develop effective quantitative methods using the same technology. These assays require state-of-the-art instrumentation and

being performed by analysts in fully equipped laboratories. Adoption of qualitative or quantitative immunochemical assays is likely to take time before

confidence and recognized legal status for such

methods is attained. It may require considerable experience and familiarization with the technology

by regulatory agencies to use test systems containing unknown reagents [“black box” test systems)

to develop procedures assuring themselves that public health protection is not compromised.

Experience and familiarization with rapid testing systems such as immunochemistry based “card

tests” is best accomplished by hands-on experience

with them and supplemented by appropriate training materials prepared by experts in theory and

technology of the rapid test systems. This is comparable to the education analysts had to acquire

when chromatography and associated instrumentation was introduced into regulatory programs.

This basic understanding enables regulatory programs and analysts to properly diagnose and evaluate test results and serves as a foundation for developing quality assurance plans and subsequent

training for regulatory control programs.

Occasionally, in the development and design of

ready-to-use products such as these tests, reliability and consistent performance of the assay from

lot-to-lot production can vary. Quality control for

production will likely improve with gained experience. Nevertheless, users of these systems must employ good quality-control and quality-assurance protocols to ensure method performance. Developing

criteria for manufacturers of such systems either

by the industry itself or by regulatory agencies planning to use such methods would be a step in the

right direction to facilitate their acceptance.

A concern facing regulatory agencies is that some

of these assays are more sensitive than the traditional quantitative and confirmatory assays, so that

these qualitative results cannot be confirmed. This

may limit further regulatory action and force technology to develop new quantitative and confirmatory methods to match the sensitivity levels. Regulatory agencies need to confirm what they have the

capability to detect, particularly at the level of interest. This level of interest is usually either an action level or tolerance established by EPA or FDA.

It is important to recognize that analytical programs designed to detect potential residue problems

must have the capability to provide quantitative

values and structure identity at or below the level

of interest. For example, within the Contamination

Response System in FSIS, an analytical result for

a pesticide or environmental contaminant that is

at or above 80 percent of the tolerance or action

level will trigger a set of specified actions, including directed sampling programs if a significant residue issue emerges. Without having the needed

quantitative and confirmatory assays to support results from a rapid test system, inappropriate regulatory actions may occur. For enforcement purposes, for residues above an established action level

or established tolerance, confirmatory methods

must be capable of unambiguously identifying the

analyte of interest at these concentrations. In situations where a tolerance or action level is established with a zero residue limit, confirmatory and

quantitative methods must work at sub parts per

million (ppm, μg/g) to parts per billion (ppb, pg/g)

concentrations based on the approved analytical

method for the analyte.

Another limitation is the heavy reliance on using

aqueous media for performing the test. For certain

food types, this may be of little consequence, but

for others it maybe a measurable deterrent. For example, most chemical-based assays rely on use of

organic solvents to release the analyte of interest

from the test sample matrix. This requires developing solvent systems providing sufficient transfer

from the organic extraction solvent to the test system while not denaturing or deactivating the immunochemical reagents. Progress is being made in

this area. For example, Immunosystems has developed an assay for chlorinated triazines (Res-I-MuneR

)

(11) that allows low rig/g (ppb) detection using select aqueous organic solvent systems. This system

is currently being evaluated by FSIS for meat

products.

Today, immunochemical assays are available not

only for chlorinated triazines but also for paraquat,

chlordane (heptachlor, dieldrin, endrin, aldrin, and

endosulfan are detected via cross-reactivity), 18

pentachlorophenol and polychlorinated biphenyls

(PCBs) at levels of interest. FSIS has method-development contracts for developing immunochemical

assays for heptachlor-related organochlorine pesticides; ivermectin; synthetic pyrethroids (permethrin, cypermethrin, and deltamethrin); and

nitroimidazoles in meat and poultry tissue (12).

These are expected to provide improved laboratory

analytical capability for these analytes. Development of qualitative screening assays is possible.

A commercial pesticide detection system based

on cholinesterase enzyme inhibition has been developed by EnzyTech, Inc. (13) The enzyme ticket
159

system detects common insecticides that account

for about 85 percent of all insecticides used in the

United States at concentrations in the low rig/g (ppb)

range. Shelf stability for the test system is estimated

to be several years. The design of the system allows

for a two-tier analytical scheme that will allow

differentiation of organic sulfur containing organophosphate insecticides from their oxygen analogs.

This advantage reduces some of the options of further analysis to quantify and confirm these analytes.

Research is being done to enable analytes from an

organic extract to be analyzed with the test system.

Development of quantitative and confirmatory analysis using other analytical technologies may be

needed to support these qualitative methods.

New column chromatography packing materials

have simplified many sample purification analysis

procedures. These solid phase extraction materials allow many solvent-to-solvent extraction and

purification systems to be eliminated from traditional methods. Future applications may become

the basis of rapid test systems requiring only solvent elution to isolate analytes of interest.

integrating Screening Methods Into

Multiresidue Regulatory Programs

Applications of screening methods for pesticideresidue regulatory programs to some extent depend

on residue violation rates. The first scenario covers instances when data indicate a low incidence

of an above-tolerance residue for approved pesticide use. The second scenario applies to situations

with a relatively higher percent of residue violation

incidence for approved pesticide use. A third scenario would be for detecting and confirming pesticide residues from unapproved pesticide use. The

first two may be influenced depending on whether

or not agreements exist for residue avoidance programs between a regulating agency and a food producer. Where such agreements are available, one

incentive to such programs would be to reduce sampling of such producers, assuming a history of good

quality-control in their production systems.

Integrating rapid test methods into regulatory programs does not imply reducing emphasis on reporting quantitative values below tolerances or action

levels. These quantitative values both below and

above these levels of interest are important for

analyzing trends and designing future residue control programs. However, integrating rapid test

methods for regulatory control programs implies

an intelligent design using rapid screening methods (commonly, Level III methods) with quantitative methods (analogous to Level II methods) and

confirmatory methods (Level I methods) to optimize

the limited resources available to regulatory control programs. With a low-level violation incidence

from statistical-based random-sampling programs,

screening methods are particularly attractive for

field or in-plant use because they allow for methods with broad versatility to test large numbers of

food products and related samples, Data indicate

that with statistical-based random-sampling programs, the large majority of samples contain nondetectable and below level concentrations of residues

(14). This provides programs with the opportunity

to clear products with non-detectable residues or

detectable below a tolerance, while retaining suspected positives for more definitive analytical procedures. This generally provides for more effective

use of expensive laboratory facilities and resources

as well as for reducing the significant costs involved

in sample collection and shipping all samples to a

designated laboratory. Data management systems

have to be appropriate to the regulatory need in all

cases.

For instances in which there is a known or highresidue violation incidence, quantitative immunochemical, enzyme-inhibition assays and thin layer

chromatographic systems designed for rapid testing in laboratory environments become very attractive. In this scenario, where large numbers of

samples are expected to give results in residue violations, the advantages to regulatory programs for

reduced analytical costs for sample collection and

shipping are diminished. The level of effort needed

for field or in-plant personnel to use and follow-up

on results from rapid tests could result in an increased workload for additional sample collection

and shipping. Using another laboratory analytical

method (another Level H method), in these situations provides an independent assay for the analyte of interest and is generally suitable for verifying initial results. This may require developing and

validating new methods using improved analytical

detectors, more sensitive color-forming reagents,

fluorescent-generating reagents, or biochemical and

color-forming reagent systems that match the sensitivity, specificity, and other screening method performance characteristics.

Another option is to allow well-defined samplecompositing schemes to be employed for laboratory

analysis. This is particularly attractive when no

known incidence of a residue problem exists. It is

somewhat less attractive under the second scenario

with known or high-residue violation rates because

it calls for reanalysis of individual samples within
160

the composite sample when an actionable finding

is indicated by analysis of the composite sample.

In the third scenario (detecting residues of unapproved pesticides or pesticide use), residue screening tests are very attractive because detection of any

amount of pesticide residue in specific products is

a residue violation. It normally requires support by

a confirmatory procedure. This assumes, as in all

other cases, performance characteristics of methods are well defined. In this scenario, quantitation

is not a specific requirement, although administrative level may be defined by an agency before initiating other regulatory action. Under any of these

scenarios, epidemiological information should be

incorporated to design effective subsequent laboratory-analysis programs. To facilitate design of an

integrated residue control program, a decision-tree

process may be a suitable objective. This will require some preliminary activities. The mission statement of the regulatory agency and the objectives

of residue analysis must be clearly defined and

adopted. Figure 1 is a suggested approach. Others

may be developed based on specific needs or other

regulatory and statutory considerations.

Consraints on Use of Rapid Test

Methods

A perceived constraint in some programs with

screening tests is that they are not specific and consume too large a portion of valuable resources to

identify the residue of interest. A possible resolution to this would be to encourage development of

1.

2.

3.

4.

References

Taylor, S., Pesticide Monitoring Program: Developing New Methods to Detect Pesticide Residues in Food, Congressional Research Service,

Apr. 24, 1987.

National Academy of Sciences, “Regulating Pesticides in Foods,” Committee on Scientific and

Regulatory Issues Underlying Pesticide Use Patterns and Agricultural Innovation, Board of

Agriculture, Washington, DC; National Research

Council, 1987,

U.S. Congress, Government Accounting Office,

Pesticides: Need to Enhance FDA Ability to

Protect the Public from Illegal Residues, GAOI

RCED-87-7, 1986, 58 pp.

Official Journal of the European Communities,

Commission Decision 87/410/EEC, July 1987.

more selective screening assays to improve the

selection process of a multiresidue or single-residue

analysis method. That is, develop a hierarchy of test

methods based on current and emerging technology, focusing on the concept of simplicity in design and application, automation technologies, and

commercially available systems and equipment

with potential for broad application.

Rapid testing systems may be compromised by

other constraints. A rate-limiting factor for application of rapid tests is, or may become, preparing

an appropriate sample for analysis. Encouragement

is warranted for research to develop improved sample preparation techniques amenable to rapid test

systems in nonlaboratory and laboratory environments. For example, supercritical fluid technology

may be a valuable research avenue for laboratory

based analysis. An alternative could be to focus on

physical or chemical properties of pesticide residues. A third possible approach would be research

on pharmacokinetic or metabolism studies to correlate pesticide residues in food and tissue matricies

to fluid extracts. Incentive is needed to encourage

commercial organizations to develop rapid test procedures. It is primarily through the economy of

scale and return on investment for manufacturers

that tests will become available and provide regulatory agencies with sufficient potential low-cost onsite, laboratory onsite, and laboratory test procedures. Development of rapid tests for regulatory use

can be fostered by offering contract funding to the

relevant regulatory agencies. Recent examples of

this method development process have shown promise, particularly with colleges and universities.

5.

6.

7.

8.

9.

10.

U.S. Department of Agriculture, New Zealand

Journal of Science, Food Safety and Inspection

Service, Revised 1987.

Sherma, J., “Pesticides and Plant Growth Regulators,” vol. 14, Modern Ana]ytica) Techniques,

G. Zweig and J. Sherma (eds.), Academic Press,

1986.

Clear, M. H., Fowler, F. R., Sony, S. R. B., et al.,

New Zealand Journal of Science 20:221, 1977.

U.S. Department of Agriculture, FSIS, Contract

53-A94-5-19, September 1985, personal communication, R. Ellis, Director, Chemistry Division,

Science Program, Washington, DC.

Lawrence, J. F., J. Assoc. of Anal. Chem. 64:758-

761, 1980,

McCausland, I., et al., Emerging Technology

For Testing Chemical Residues in Meat, Report

of Australian Meat and Livestock Research and
161

Figure I.-Decision Tree for Regulatory Control Program

Develop regulatory

mission statement

and objective

Design regulatcry program

I Conduct testing to I

Insufficient or no determine residue incidence

residue incidence

No Low need for field screen

High need for field screen

incidence incidence

/

r t \

Yes Implement screen test Yes

for field/plant use

*

Conduct field test

Negative

I

Positive

)

Implement screen test *

No for laboratory use

I

t

Conduct lab test

Positive Negative

* /

1 I

f

Yes

At or above

Below level

of interest

I

level of interest Perform quantitative

analysis

Perform confirmatory

analysis

Confirmed Not confirmed Review for additional m program analysis

Take appropriate

L

regulatory action

SOURCE: Richard EIIIs, Food Safety end Inspection Service, U S Department of Agriculture, 198S
162

Development Corporation, Sydney, NSW, Aus- 13. EnzyTec, Inc., 8805 Long, Lenexa, KS 66215,

tralia, October 1987. Product Bulletin, Pesticide Detetor Ticker.

11. ImmunoSystems, Inc., 8 Lincoln Street, P.O. 14. U.S. Department of Agriculture, Food Safety

Box AY, Biddeford, ME 04005. and Inspection Service, Domestic Residue Data

12. R. Ellis, Director, Chemistry Division, Science Book National Residue, 1985, May 1987.

Program, U.S. Department of Agriculture, FSIS,

Washington, DC, personal communication.
163

The Role of Robotic Automation in the Laboratory

Bruce E. Kropscott and Cynthia N. Peck, The Dow Chemical Company, Midland, MI, and

Harry G. L ,

Abstract . . . . . .

Introduction . .

Campbell Institute for Research and Technology, Camden, NJ

Contents

Page

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

Robotics Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..............164

Advantages of Robotic Automation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..164

Constraints of Robotic Automation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..165

Key Elements to Implementation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ..166

Robotics Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................166

Current Status of Automation in Pesticide Residue Testing . ....................167

Constraints to the Use of Robotics for Multiresidue Testing . ...................168

Future Prospects for Automation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..............168

Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................169

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..”.”””.””””””. , ,169

Acknowledgment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........................169

Other Sources of Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............170

Abstract

Rapidly increasing numbers of samples for pesticide residue analysis have forced the analysts in

analytical laboratories (governmental, industrial,

and private) to look for ways to increase sample

throughput. This trend has provided a need for the

development of a variety of automated equipment

for the analytical laboratory.

In support of the workshop on ’’Technologies to

Detect Pesticide Residues in Food’’ sponsored by

the Office of Technology Assessment (OTA), this

paper will l) provide an overview of laboratory automation, 2) assess the emerging robotic technology

for the analytical laboratory, 3) review the current

status of automation in pesticide residue analysis,

and 4) identify research needed for expanded automation in the analytical laboratory.

Introduction

Scarcely an aspect of modern life has be enunaffected by automation, yet defining automation is

difficult without using automatic or automated in

the definition. Consider Webster’s definition: “the

technique of making an apparatus (as a calculating

machine), a process (as of manufacturing), or a system (as of bookkeeping] operate automatically” or

the condition of being automated(l). Automation

implies the integration of a self-governing system.

Unlike mechanization, which is defined as simple

replacement of human labor by machines, true automation must have the ability for feedback control

and the ability to regulate. Four key elements of

automation are 1) a source of power, 2) sensing

mechanisms, 3) decision elements, and 4) control

elements (2). For all practical purposes, laboratory

automation is the use of devices that perform repetitive tasks. Reviews of large annual trade and equipment shows, such as the Pittsburgh Conference &

Exposition on Analytical Chemistry and Applied

Spectroscopy, provide an excellent overview of current automated equipment.

Laboratory automation can be divided into four

basic categories:

1) Dedicated, single-task

2) Dedicated, multi-task

3) Computers

4) Robotics

The simplest type of automation is a dedicated,

single-task instrument. These instruments may be

commercially available or custom manufactured

and perform just one independent function. Examples of single-task automated equipment include

autoinjectors, electronic balances, timed shakers,

centrifuges, switching valves, etc. Dedicated, singletask devices are well-established and widely used

in most laboratories.

The next category of automation is the multi-task

device. Instruments in this category perform multiple tasks such as diluting, mixing, filtering, solid

phase extraction, chromatographic separations, etc.
164

Examples of dedicated multi-task automation include automated sample preparation devices that

combine adding solvents, mixing, extracting, filtering, evaporating, etc. and multiple-step auto samplers that can heat or cool, add internal standards

or derivating agents, serially dilute, etc. Most dedicated multi-tasking devices are highly specialized.

For example, automated cleanup and extraction devices facilitate rapid processing of a highly selective number of repetitive, routine samples but remain

subject to obsolescence and represent isolated,

stand-alone equipment. The number of commercially available, multi-tasking devices is markedly

less than its single-tasking counterpart. Additional

multi-tasking devices include hyphenated technologies such as gas chromatography-mass spectrometry (GC/MS), liquid chromatography-mass spectrometry (LC/MS), and gas chromatography-infrared

spectrometry (GC/IR), etc., which couple established, analytical technologies to enhance the automated detection and measurement of compounds.

Computers differ from the first two types of automation since they automate data handling and calculations instead of physical and mechanical manipulations. This category of automation includes

hand-held calculators, integrators, personal computers for control and data handling, and networked

laboratory information management systems (LIMS).

Computers and associated microchip technology

have developed into a modern “Industrial Revolution” that is extremely vast and important but beyond the scope of this paper.

Robotics evolved as hybrid systems incorporating technology from both mechanical and computer

(microprocessor) automation. Robotics uses a computer-controlled, mechanical manipulator to incorporate both single- and multi-tasking automated

devices into an integrated system. Since its commercial introduction and implementation in the

early 1980s, laboratory robotics has provided

reprogrammable, multi-tasking, computer-assisted

automation in the laboratory for a variety of pesticide and non-pesticide applications. The cost of a

laboratory robotics system typically ranges from

$40,000 to $60,000. The return on investment depends upon the application but is typically 6 months

to 3 years. Before discussing robotic automation,

a working definition of a robot must be established.

A definition of a robot is in itself a formidable

task, since there are so many misconceptions and

preconceived ideas of what a robot is. A definition

of a robot that has been adapted from the Robotic

Industries Association’s definition is as follows: A

robot is a reprogrammable, multifunctional manipulator designed to move materials, parts, and specialized devices through a variety of computercontrolled motions for the performance of a variety of tasks (3). The key words are reprogrammable, multifunctional, and computer-controlled.

Misconceptions and preconceptions of robots are

difficult to overcome. Most every equipment manufacturer wants products that are associated with

the latest technology. An example of equipment that

does not fit the definition is a “robotic” autosampier. The autosampler transports vials from a tray

to an injector with precise timing, performs multiple injections, and varies the size or speed of injections. The autosampler is not a robot: it is an automated instrument that performs a single-task of

repetitive, precision injections. In addition, most

toys or mechanized trade-show mannequins do not

fit this definition of a robot.

Several types of robots that do fit the definition

include the following: 1) industrial, 2) research and

development, and 3) laboratory robots. The first

industrial robot in the United States was sold by

Unimation to General Motors in 1961. Currently,

industrial robots perform such tasks as welding,

painting, parts assembly, and material handling. Research and development robots cover such areas

as education, cybernetics, and space exploration.

In 1982, another type of robot was introduced, a

laboratory robot. Its major function has been automated sample preparation. The remainder of this

paper will limit its scope to assessing the emerging

technology of the laboratory robot, discussing its

advantages, constraints, current applications, and

future prospects.

Advantages of Robotic Automation

The advantages of robotics include many of the

same advantages as conventional automation and

are summarized in five categories:

1) cost-effectiveness

2) reproducibility

3) versatility

4) safety

5) automated documentation of procedures.

Robotics can be cost-effective. Like conventional

automation, robotics provides a competitive advantage in that the robots can work extended hours and

increase the use of existing equipment. Robots free

personnel from performing repetitive tasks and

devoting constant attention to minute details, thus
165

allowing additional work or research to be performed. The robot is not subject to hunger, boredom, fatigue, or illness. Robots do not need promotions, pay raises, or medical benefits. However,

robotics does not preclude the need for well-trained,

competent analysts.

Reproducibility is a major attribute of a robotics

system. Performing tasks with the exact timing and

uniform sample handling, the robotics system separates the actual chemistries involved from variations in techniques, which may in turn lead to improved precision and accuracy in methodology.

This is very useful in method validation, where conditions and parameters can be systematically varied to optimize new methodology during the validation process. Transfer of robotics technology

could improve intra- and interlaboratory reproducibility.

Robotics is versatile automation and thus less subject to obsolescence. The reprogrammability of the

robotics system allows method optimization or a

complete change of application to meet changing

needs in a laboratory. Robotics technology also

bridges the gap between what is commercially available in dedicated automation and what is actually

needed for a specific application (e. g., custom sample preparation, instrument interfacing, specialized

autosamplers, etc.).

The use of robotics reduces human exposure to

hazardous chemicals, extreme temperatures, and

other undesirable conditions such as pinch points,

defective glassware, and sharp objects.

Finally, the computer portion of the robotics system can provide automated documentation of the

procedure. The entire program as well as sample

weights, dilutions, timing, calibrations, etc., can be

printed or transferred directly to a host computer,

vastly reducing transcription errors. Computerized

documentation thereby can establish an audit trail

for the entire procedure.

The present technology of laboratory robotics has

several constraints, which are grouped into the following categories:

1) New technology

2) Mechanical and computer failures

3) Spatial and physical limitations

4) Safety

5) Associated technology lagging behind automation

Laboratory robotics is still an emerging technology.

The world market for laboratory robotics in 1985

was estimated at 30,000 to 50,000 units (4), but this

market has not been reached because there seems

to be a general reluctance to change and a lack of

wide acceptance of any new technology. As of December 1987, laboratory robotics systems numbered

about 1,3oo, and only a limited number of personnel were trained in the operation and repair of robotics systems. To date, Zymark Corporation has

more than 85 percent of the present laboratory robotics market. Other laboratory robotics companies

include Lerkin, Fisher Scientific, and Hudson Robotics.

The majority of robotics systems have required

significant cost and time to be fully programmed

and functionally implemented. Newer robotics systems, such as the PyTechnology introduced by Zymark Corporation in 1986, have reduced start-up

cost and time by providing systems that are preprogrammed and pre-positioned for basic laboratory operations.

Robots are hybrid systems that combine mechanical equipment with computers. This results in a

combination of problems associated with machines

(such as physical wear, mechanical failure, etc.) and

with computers (electrical power, noise spikes,

“glitches”).

Spatial constraints also limit the robotics system

because the robot is confined to its working envelope, typically less than a cubic meter. Exact positioning, spatial orientation and readily accessible

work areas are necessary for the robot to interact

with peripheral modules and support equipment,

Modules and equipment are usually bolted on the

table or placed in a rack to assure proper positioning. As a result, samples and solvents are limited

in size (0.1 to 50 ml) and weight (less than 3 lbs.)

to maximize the use of the working envelope.

A human has extensive systems of intricate sensors and feedback mechanisms that provide information about the environment (e.g., touch, pressure,

temperature, hearing, sight), A human also possesses an extensive memory from which to recall

and process that information. In comparison, a robot has a limited memory, limited feedback mechanisms, limited dexterity, and limited artificial intelligence. Thus safety, which was listed as one of

the advantages of robotics, can become a liability

when errors occur that require logical decisionmaking to recover from an unanticipated situation for

which it was not designed or programmed,

Associated technologies (disposable supplies,

glassware, ancillary equipment, etc.) are lagging behind the robotics technology. The associated technologies are not ready for a blind analyst with
166

limited dexterity (4 to 5 degrees of freedom compared with more than 40 degrees of freedom in the

human upper limb). A robotics system must be

viewed as an integrated system consisting of the robotic arm, peripheral work-station modules (such

as vortex mixers, centrifuges, evaporators, etc.) and

supporting equipment (such as test tubes, pipet tips,

extraction columns). Since robotics systems are integrated systems, they are only as good as the

weakest component. Modules with moving parts

such as shakers, centrifuges, and vortex mixers

need to be manufactured for computer control and

must be designed to return to an exact position to

compensate for the blind analyst. Disposable items

(such as pipet tips that are bowed, screw-capped

bottles that will not seal, etc.) can produce spills,

malfunctions in laboratory operations, or in the

worst case, cause catastrophic failure in unattended

operations. Although these types of laboratory supplies have been around for years, the specifications

and quality control in their manufacture did not become an issue until they were used in conjunction

with robotics systems. A failure is a failure whether

it is caused by a $2 tip, a $7 centrifuge tube, or a

$50,000 robotics system.

At the Third International Symposium on Laboratory Robotics, Frank Zenie, president of Zymark

Corporation, summarized the key elements to implementation of robotics. “Once adequate funds and

people are available, the following four requirements are key to all successful automation projects:

1) motivated people, 2) proven chemistries, 3) disciplined planning, and 4) creative implementation”

(5).

Robotics is an emerging technology and as such

needs development from key, dedicated personnel.

The technology is new enough that systems will fail

from short-term problems (lack of time, lack of resources, lack of key people). There are many obstacles that can make a robotics system fail, but with

motivated, dedicated people these problems can be

overcome.

Automation without valid, proven chemistries is

useless. If the application is not based on sound,

reliable chemistry, robotics systems will only be automating the generation of meaningless numbers

and useless results. On the other hand, robotics with

its exact timing and uniform sample handling can

be used as a research tool for separating the chemistries involved from the variability in the manual

techniques.

Disciplined planning is another key element in

successful implementation. Robotics systems have

a great amount of versatility but actual applications

are well-defined and limited in scope. The goal,

tasks, and laboratory unit operations (LUOs) need

to be well defined and focused to allow automation,

Examples of well-defined applications versus openended applications are as follows:

Well-defined versus Open-ended

machine, tool and sculpting

die; writing a cal- writing poetry

culations program; basic research

production of

goods;

analysis of organo- versus determination of

chlorines in corn all pesticides in all

using the Luke food groups

screening method

Creative implementation is necessary since the

robot can not emulate the human in task performance. “The analogy between the marvelously dextrous human hand and the robot hand is extremely

crude, as is the analogy between human learning

and robot programming” (6). The good news is that

the robot does not have to emulate the human in

task performance. The human hand has more than

50 distinctively different movements. That degree

of dexterity is not necessary to pick up a test tube

and move it to a balance, mixer, etc. In developing

conventional laboratory methodology, humans incorporate unit operations that maximize their

strengths and minimize their weaknesses. As a result, manual methods are validated with procedures, timing sequences, and specific laboratory

tools that are most efficient and convenient for the

human. If a manual application is to be performed

by a robot, tasks must be modified, then optimized,

and finally programmed for the robot’s capabilities.

Robotics systems can be used for automating method

validation as well as for routine sample preparation. Methods can be developed and optimized by

systematically varying parameters, eliminating the

need for first developing manual methods that need

to be modified for use with robotics.

Robotics Applications

Laboratory robots and workstations have automated a variety of laboratory unit operations (LUOs)

including weighing, pipetting, diluting, filtering,

centrifuging, evaporating, solvent dispensing, mixing, etc., which can be sequenced for specific applications. More than 100 applications have been

developed in the pharmaceutical, chemical, biologi-
167

cal, environmental, biotechnical, and food industries.

More than 45 percent of those robotic applications

are in the pharmaceutical industry. Currently more

than 200 companies in the United States are using

laboratory robotics. As of December 1987, the seven

largest customers of laboratory robotics were The

Dow Chemical Company, DuPont, Eastman Kodak,

Eli Lilly, Merck, Monsanto, and Procter & Gamble,

totalling almost 20 percent of the current robotics

systems (7).

Current Status of Automation in

Pesticide Residue Testing

The presence of several hundred registered pesticides has led to the development of multiresidue

screening procedures. The FDA’s pesticide analytical manual (PAM) describes screening procedures

that are distinguished by both the type of food group

and type of pesticide residue that are probably

present. [8] All these methods have four basic, common operations: 1) obtaining a representative sample, 2) sample cleanup, 3) chromatographic analysis, and 4) data reduction and reporting.

Methodologies begin with procurement of a representative food material, preparation (peeling,

grinding, homogenizing, etc.), and extraction with

an organic solvent (acetonitrile, acetone, petroleum

ether, etc.). The extraction step is usually a manual

operation with large variations in sample and solvent sizes depending upon the food and levels of

pesticides. This step is very labor-intensive, timeconsuming and difficult to automate.

The sample cleanup isolates the pesticide from

the rest of the matrix. This may be accomplished

by techniques such as liquid/liquid partition, gel

permeation chromatography, or solid phase extraction (SPE) columns (i.e., Florisil columns).

Sample cleanup is very labor-intensive and timeconsuming. Developments in automating sample

cleanup have been reported by several authors. For

example, Stallings et al. applied automated gel chromatographic cleanup to the analysis of pesticides

in fatty-food materials (9). Gretch and Rosen reported a cleanup procedure for multiresidue testing using automated solvent partitioning extractions interfaced with a column chromatography

module for automated solvent partitioning extractions (10). Other automated gel permeation and

chromatography systems addressed the cleanup step

by automating collection of eluent fractions, evaporation, and solvent substitution as well as injection

into a gas chromatography (11, 12). A review of papers

from the Advances in Laboratory AutomationRobotics indicated robotic automation of the

cleanup step using solid-phase extraction (SPE)

columns of a variety of pesticide and non-pesticide

compounds from a variety of matrices (13, 14, 15).

The third step involves the actual chromatographic

analysis. The three most common techniques are

gas chromatography (GC), liquid chromatography

(LC), and gas chromatography-mass spectrometry

(GC/MS). Types of gas chromatography detectors

used for pesticide residue analysis include electron

capture, flame ionization, or Hall conductivity.

High performance liquid chromatography (HPLC)

with ultraviolet or fluorescence detection is also

widely used for pesticides that are not readily analyzed by GC. Autosamplers coupled with chromatographic instrumentation facilitate the automation

of the analysis step.

Finally, the data reduction and summary reporting step has been largely automated with the use

of computers,

Due to the newness of automation technology and

the timeframe required for method development,

review, and publication, the literature does not reveal much in the way of published information on

the specific application of robotics to multiresidue

testing. It is very difficult to breakdown the percentage of work that is being done using automation

versus manual preparation since there is so much

variation from laboratory to laboratory in the type

and amount of equipment, funding, and personnel.

Private inquiries as to the status of automation by

the authors of this paper indicated a considerable

amount of research being done.

Automation of preparation, cleanup, and detection of pesticide residues in foods has typically focused on single, discrete operations. Specific tasks

have been automated with single-task, dedicated

equipment such as autoinjectors, which make repetitive, precision injections into chromatographic instruments. Reducing the variables in injection techniques allows more unattended operations and

higher use of the analytical equipment. Automation

in the final analysis step with the use of autosamplers and computerized data systems was a major

accomplishment in increasing sample throughput

in the analytical laboratory and has been incorporated in most of the laboratories, yet it did not address the labor-intensive extraction or cleanup.

Dedicated, multi-task instruments have been developed for automated fraction collection, and solvent

exchange can process several dozen pesticide residue samples sequentially. The $20,000 to $40,000

capital investment has prevented the incorporation

of this equipment into some laboratories. Laboratories with large numbers of the same sample type
168

and limited personnel resources may derive the

most benefit from dedicated automation. On the

other hand, a $50,000 robotic system may provide

a better investment for these laboratories since the

flexible, automated system may be used to process

different types of samples as needed to meet the

varying demands and optimize current methodology. As pesticide technology changes, the system

can be upgraded to avoid obsolescence and reprogrammed to meet changing analytical needs.

At the Third International Symposium on Laboratory Robotics, Tinier et al. reported on their determination of deltamethrin (active ingredient in

Decis insecticide) and its metabolize in milk and

vegetables at the 50 ppb level (16). Their robotics

system was used as a tool for optimization of various LUOs that are commonly used in screening

techniques such as centrifugation, multiple solvent

extraction, mixing, and solid phase extraction

cleanup.

Applications using robotics for the automation

of the Luke multiresidue screening procedure has

been recently reported by Grady and Lento (17). Test

results on several food matrices (tomatoes, corn,

peas, and carrots) using several different organic,

chlorinated pesticides gave similar results to those

obtained by the manual assay. They predict continuing developments in the application of robotic

techniques to other types of multiresidue screening tests, particularly in assays of fatty-type foods,

organic phosphates, and methyl carbamates.

Constrains to the Use of Robotics

for Multiresidue Testing

Regulatory and environmental samples are not

readily automated because of some of the following factors:

1) Diverse type of samples and matrices

2) Widely varying classes and concentrations of

pesticides

3) Large quantities of samples and reagents handled

4) Varying complexity and multiple sequences of

extraction, cleanup, analysis

5) Lack of large series of similar samples

6) Need to show equivalency to official methodology

Samples requiring residue analysis have many

variables (constraints 1-5), which interfere with the

total automation process. In addition, many present

day procedures were developed using equipment

that is not suitable or compatible with other types

of automation. The research cost for the development of new equipment to replace existing equipment is also a formidable problem.

The need to show equivalency to established

methodology has placed a serious constraint on the

practicality of automating current methods. Often

it is cheaper for companies to stay with the manual

techniques than to spend the time and effort on

evaluating and implementing any new automation.

Efforts need to be continued in the government and

private industry to research and develop automated

technology, particularly in the following areas:

1) robotics

2) laboratory information systems

3) automated cleanup apparatus

4) new, specific detectors

5) artificial intelligence/expert systems

Existing techniques and equipment need to be networked and integrated into working systems, not

used solely as isolated workstations.

To test specifically for each of several hundred

pesticides registered for use would be costly and

extremely inefficient. Adoption of screening techniques (which could be automated) in conjunction

with official methods (used for specific confirmation of over-tolerance samples) could allow for a

rapid throughput of large numbers of samples without sacrificing regulatory methodology. The small

number of suspect samples maybe reanalyzed using

the more lengthy, time-consuming, and specific

methodology. A tolerance assessment system should

be maintained with methodology that is shorter and

instrumentation that is sensitive and specific. By

narrowing the scope of the problem (i.e., from measuring for all types of pesticides in all types of foods

to development of a series of screening techniques

for specific classes of compounds in specific groups

of foods), the possibility of automation then becomes

more a reality.

Once a method is modified for the robotic system, subsequent method validation is not a constraint due to the exact timing, uniform sample handling, and reproducibility that is inherent in robotic

systems. Transfer of robotic technology can easily

be accomplished, making the multi-validation procedure required for official validation easier.

Future Prospects for Automation

Future prospects for robotic automation include

improvements in the following areas:

1) robotics

2) computers

3) sensors

4) associated technologies

Although pre-programmed robotics technology is

still not fully developed, advances in that technol-
169

ogy could have the same positive impact that preprogrammed software packages had for the microcomputer systems, allowing an analyst to use the

instrumentation without requiring an extensive

knowledge of programming or theory of operation.

Research and development is needed for improvements in existing electronics and mechanics of the

robot. Expanded sample and solvent sizes (micro

to macro-semi prep) are also needed for a variety

of robotic applications.

Advances in computer technology will improve

robotics, instrumentation, and data handling. The

robots will need more memory, auxiliary control

functions, graphics software, smaller physical size,

and fewer hardware/software problems. Instrumentation must be able to communicate with computers

as well as other dissimilar equipment without the

need for extensive programming or additional interface modules. Computer technology must allow

further integrated, networked automation in the laboratory.

Sensory technology such as sight, touch, hearing,

temperature, and pressure need to be developed,

miniaturized, and enhanced for the laboratory robot. Research and development of sensors will aid

in the safety, performance, and feedback mechanisms of the new robotics systems.

One of the biggest opportunities for future improvement in robotics technology is with the associated technologies. Providing improved quality

control and new designs in disposable (glassware,

plasticware, etc.) can reduce the potential errors in

unattended operations. Research and design of specific workstations (such as cappers, mixers, evaporation stations, etc.) that can be easily implemented

with the existing robotics architecture will provide

new application opportunities for the automated

analyst.

“Laboratory automation is not based on a single

technology, but rather on several technologies that

can be focused on different parts of a lab operation.

Some of those approaches are mature, others are

evolving, and others are still experimental. Thus you

should not attempt to implement a system in one

grand stroke, but rather consider the options and

plan a stepwise implementation” (8). If an advantage is to be gained using automation, all facets of

the methodology must be examined. For example,

while great strides have been made in automated

analysis and data reduction, the sample preparation still involves much manual labor in many of

the laboratories surveyed. Reviewing the prospects

for future automation, several factors must be considered before choosing automated equipment such

as amount of funding, training, and availability of

personnel; numbers and types of current and anticipated samples; and where the strategic advantage would be gained using a particular type of automation.

Summary

Robotics allows reprogrammable, multifunctional,

computer-controlled automation for a variety of laboratory unit operations up to and including complete applications. Although robotics is an emerging technology, it has made great strides in

laboratory automation by addressing the need to

link isolated workstations with one another and has

provided a means for an integrated, laboratory

network with other automated systems. Robotics

should continue to’ develop as a fundamental, integral tool for sample preparation, automation, and

research and development in analytical laboratories.

Acknowledgment

The authors thank the following people and their

companies for contributions to this paper in laboratory robotics: Albertha Paul, Zymark Corp.; William Kruka, Perkin-Elmer; Raymond Reilly, Hudson Robotics; Terry Hight, Radian; Ashok Shah,

Fisher Scientific; and Frank McCullough, ABC Laboratories.

References

1. Webster’s Third New International Dictionary

(Springfield, MA: G&C Merriam Inc., 1981).

2. New Encyclopedia Britannica, vol. 1 (Chicago,

IL: Encyclopedia Britannica Inc., 1987).

3. Conlan, Roberta (cd.], Robotics: Understanding

4.

5.

6.

7.

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(Washington, DC: Food and Drug Administration, 1981).

9. Stallings, D, L., Tindle, R. C., and Johnson, J, L.,

J. Assoc. Off. Anal. Chem. 55:32-38, 1972.

Computers (Alexandria, VA: Time-Life Books,

Inc., 1986).

Freifeld, K. “The One-Armed Chemist,” Forbes,

135 (9) April 29, 1985.

Zenie, F., “Strategic Trends in Laboratory Automation-1985, ” Third International Symposium

on Laboratory Robotics, Boston, October 1985.

Ullrich, R.A., The Robotics Primer (Englewood,

NJ: Prentice-Hall, Inc., 1983).

Paul, A., personal communication 1/88, and Zymark Corporation—A Brief History, 12/4/87.

Pesticide Analytical Manual, vol. 1, 2nd eel.
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16.

Gretch, F. M., Rosen, J. D., “Automated Sample

Cleanup for Pesticide Multiresidue Analysis III.

Evaluation of Complete System for Screening

Subtolerance Residues in Vegetables,” J. Assoc.

Off. Anal. Chem. 70 (1):109-111, 1987.

Johnson, J. J., Sturino, E. E., and Bourne, S. “An

Automated Gas Chromatographic System for

Pesticide Residue Analyses,” EPA, 905/4-77-001.

Hopper, M. L., Griffitt, K. R., “Evaluation of an

Automated Gel Permeation Clean-up and Evaporation Systems for Determining Pesticide Residues

in Fatty Samples,” J. Assoc. Off Anal. Chem.

70(4): 1987.

Advances in Laboratory Automation-Robotics

1984, Zymark Corp., Hopkinton, MA, p. 61,71,

and 105, 1984.

Advances in Laboratory Automation-Robotics

1985, Zymark Corp., Hopkinton, MA, p. 111,

131, and 465, 1985.

Advances in Laboratory Automation-Robotics

1986, Zymark Corp., Hopkinton, MA, p. 37, 71,

291, 451, and 595, 1986.

Tinier C., Allegret, H., and Devaux, P. “Applications of Robotics in Residue Analysis, ” Advances in Laboratory Automation-Robotics 1986,

Zymark Corp., Hopkinton, MA, pp. 291-311,

1986.

17. Grady, M. and Lento, H., “Application of Robotics to Multiresidue Testing in Foods,” Pittsburgh Conference and Exposition on Analytical Chemistry and Applied Spectroscopy, New

Orleans, February, 1988.

18. Liscouski, J., “Issues and Directions in Laboratory Automation,” Anal. Chem. 60(2), 95-99,

1988.

Other Sources of Information

Osborne, David M., Robots-An Introduction to

Basic Concepts and Applications, Midwest SciTech Publisher, Inc. (Detroit, MI: 1983).

Krasnoff, Barbara, Robots: Reel to Real (New York:

Arco Publishing, Inc., 1982).

Reichardt, Jasia, Robots: Fact, Fiction, and Prediction (New York: Viking Press, 1978).

Joseph, Michael, The Timetable of Technology (London: Marshall Editions Limited, 1982).
171

Potential of Immunoassay in Monitoring

Pesticide Residues in Foods

Ralph O. Mumma, Department of Entomology, Pesticide Research Laboratory, The Pennsylvania State University, University Park, PA

and

Kenneth W. Hunter, Jr., Biotronic Systems Corporation and Westinghouse Bio-Analytic

Systems Company, Rockville, MD

Page

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ...171

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...................171

Immunoassay for Agrochemical Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ...172

Preparation of Anti-Chemical Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ...172

Hybridomas and Monoclinal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ....174

Principles of Chemical Immunoassay . . . . . . . . . . . . . . . . . . . . . . . . . . . ..........174

Practical Applications of Chemical Immunoassay . ..........................176

Real-Time, On-Line Agrochemical Monitoring . . . . . . . ........................178

Comparison of Immunoassays With Conventional Methods

of Pesticide Residue Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .178

Conventional Methods Versus Immunoassay Methods. . . . . . . . . . . . . . . . . . . . . . . .178

Advantages and Disadvantages of Immunoassays . ...........................179

Constraints, Opportunities, and Recommendations . ...........................179

Regulatory Agencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................179

Legislative Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......................180

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..180

Figures

Figure Page

l, Antibody Affinity and Specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...........173

2. Monoclinal Antibody Preparation. . . . . . . . . . . . . . . . . . . . . . ...................174

3. Applying Reagents to 96-well Plate . . . . . . . . . . . .............................175

4. Competitive Inhibition Enzyme Immunoassay . . . . . . . . . . . . . . . . . . . . . .........176

5. Standard Curve: Parathion Immunoassay . . . . . . . . . . . . . . . . . . . . . . . . ..........177

6. Computer, Printer, and Spectrophotometer . . . . . . . . . . . . . . . . . . . . ., , . . . , ., , ., .177

7. Generalized Biosensor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ....178

The principles of immunoassays are presented,

and selected applications of these assays for analysis of pesticide residues are cited; the advantages

and disadvantages of immunoassays are compared

with conventional analytical methods. The constraints and opportunities of immunoassay are

discussed in light of regulatory and legislative influences.

Analysis of agricultural commodities for agrochemical residues is usually time-consuming and

performed by highly skilled chemists utilizing expensive analytical equipment. Consequently, costs

of analyses are high. Five commonly used multiresidue methods detect 203 different pesticide

residues, but this is less than half of the pesticides

that the Food and Drug Administration (FDA)
172

claims may occur in foods. A number of the pesticides not detected in the multiresidue methods are

classified as high health hazards and must be analyzed by single residue methods. Again, the cost of

analysis is a major limiting factor in how many analyses can be performed. Regulatory agencies responsible for ensuring the safety of agricultural commodities, both grown at home and abroad, do not

have limitless finances and can only analyze a fraction of the samples that should be analyzed, A small

percentage of samples possess illegal levels of pesticides, but unfortunately, we have to analyze all

the samples to determine the few bad ones. Not only

are Federal and State regulatory agencies concerned

about pesticide residues, but the general public is

taking an ever-increasing interest and demanding

greater assurances of the safety of food and water.

United States’ farmers also have raised questions

about the importation of agricultural products from

countries that fail to regulate pesticide usage, for

the use of high levels of pesticides can certainly increase a farmer’s yield per acre and provide a competitive edge in the marketplace.

There is a need for simple, rapid, cost-effective

screening techniques for pesticide residues in raw

and processed foods. Since most foods do not contain illegal residues, inexpensive semiquantitative

techniques could screen large numbers of samples,

and only those few samples found in violation could

be further analyzed by more conventional means.

Immunoassay offer many of these advantages.

They have been routinely used for many years in

clinical and forensic laboratories for analysis of

small molecules such as hormones and drugs. The

procedures are becoming so simple that they are

now being conducted in doctors’ offices and even

in private homes (e.g., pregnancy tests). Immunoassay should be equally applicable for the analysis

of pesticide residues.

This article will briefly introduce the principles

of immunoassays, present selected applications of

these assays for analysis of pesticide residues, and

compare the advantages and disadvantages of this

technique to conventional analytical methods. We

will also discuss our prejudiced views of the constraints and opportunities for adoption of these

techniques and how the regulatory and legislative

branches of government can and do influence evaluation and acceptance of immunoassays.

Immunoassay for Agrochemical

Analysis

The use of antibodies to identify and quantify agricultural chemicals grew out of the clinical use of

antibodies in infectious disease diagnostics and

therapeutic drug monitoring. The immunologic

principles behind the technology have been known

for some time, so the relatively late onset of antibodybased analysis of agrochemicals was probably due

to a failure to recognize its potential outside the

medical arena. Even after the publication of the first

chemical immunoassay in the scientific literature,

no commercially available or regulatory agencyapproved chemical detection system based on antichemical antibodies was seen until recently.

Preparation of Anti-Chemical Antibodies

Antibodies are produced and secreted by plasma

cells, the end-stage differentiated cell of the Blymphocyte series. Plasma cells can be thought of

as antibody-producing factories. The immunological

rule of “one cell, one antibody, ” means that only

one kind of antibody is made and secreted by one

plasma cell (27). The antibody molecule evolved as

one of an animal’s major lines of defense against

foreign substances such as pathogenic microbes.

Antibodies are proteins whose primary amino acid

sequence dictates a tertiary or three dimensional

structure that bears a site into which a distinct

chemical structure can bind (26). Some of the structures that can induce and interact with antibodies

include sugars on the capsules of bacteria, viral

glycoproteins, or glycoopids on tumor cells, but

nearly any chemical structure, if presented to the

immune system in the proper configuration, can

induce and bind with a particular antibody. The

analytical capabilities of antibodies have been appreciated for a long time (2, 17). Indeed, to measure the amount of a particular substance, one can

inject the substance into an animal, isolate the antibodies to the substance, and in one of many modifications of immunoassay, detect and quantify the

substance. Such is also the case with antibodies to

small organic chemicals, but another tenet of immunology must first be considered before these

chemicals can induce antibodies. The small size of

nearly all agrochemicals forestalls their ability to

induce the production of anti-chemical antibodies.

Nevertheless, by attaching the small chemical to a

larger immunogenic carrier molecule such as a foreign protein, the immune system of an animal can

be coerced into producing an anti-chemical antibody. A chemical structure too small to induce an

antibody by itself, that when conjugated to a larger

carrier molecule induces a specific antibody, is

termed a hapten (17).

When injected into an animal, a chemical haptencarrier complex induces an array of different anti-
173

bodies. There are antibodies to the carrier molecule

in abundance, antibodies that bind to the haptencarrier combination, and antibodies that bind only

to hapten. It is only those antibodies that recognize

the haptenic structure alone that are of value for

the development of immunoassay, However, the

antibody response to a particular hapten is extremely heterogeneous. Again, another tenet of immunology is based on the clonal selection theory

(5). This theory states that foreign substances, such

as the haptens, do not instruct the immune system

to manufacture an antibody with structural complementarily to the hapten. Rather, there pre-exists

in a mammalian immune system a B lymphocyte

that is programmed to produce and secrete, upon

stimulation by the proper chemical structure, an

antibody with binding affinity to that structure, In

essence the hapten selects from a pre-existing repertoire of B lymphocytes. In a typical immune response to a hapten, even with the limited size of

a typical haptenic molecule, dozens or even hundreds of B lymphocytes with surface receptors capable of interacting even weakly with the hapten

are stimulated to undergo proliferation and subsequent differentiation into end-stage, antibodysecreting plasma cells. Each of these clones of

plasma cells secretes an antibody that recognizes

in some way the haptenic structure. The family of

antibodies that accumulate in the plasma of an animal following immunization with hapten-carrier are

termed polyclonal in that they issue from many

clones of plasma cells. The serum of such an immunized animal can be used as a source of antichemical antibody, and this antibody can be manipulated in ways to be discussed later for the quantification of the chemical.

The property of antibody binding to a chemical

can be described by two closely related terms. The

first is affinity, a term used to describe the strength

of the interaction between chemical and antibody.

Affinity is determined by the sum of all operant noncovalent chemical interactions (i.e., hydrogen bonds,

van der Waals forces, hydrophobic and electrostatic

interactions). A schematic representation of affinity

is shown in figure 1. In this illustration, three antibodies are shown, all of which bind to the hypothetical chemical. The one on the left shows a perfect

fit with the chemical and is thus a high affinity antibody. The middle antibody has one region of noncomplementarity and is therefore of medium affinity, and the antibody on the right has only one

complementarity region and is described as low affinity. The concept of affinity is important because,

for most immunoassays, the higher the affinity of

Figure 1. —Antibody Affinity and Specificity

ANTIBODY AFFINITY

High Mediu m L o w

Affinity Affinity Affinit y

ANTIBODY SPECIFICITY

Specifi c Cross- Reactive Non - Reactive

SOURCE: Ralph Mumma, Pennsylvania State University and Ken Hunter, West.

inghouse Bio-Analytic Systems, Co., 1988.

the antibody for the target chemical, the greater the

sensitivity of the immunoassay.

The specificity of antibody-chemical interactions

is illustrated in figure 2. Specificity and affinity are

closely related terms, but they can be differentiated

for illustration. Here we have one antibody and four

chemical structures. On the left is a very specific

interaction between antibody and chemical, and on

the far right there is no interaction at all. The two

chemicals in the center show a degree of crossreactivity with the antibody due to a degree of structural homology with the chemical on the left. Specificity is of considerable importance because an immunoassay, as any analytical method, must be able

to distinguish between related chemicals.

As mentioned earlier, the serum of an immunized

animal can serve as a useful source of anti-chemical

antibodies for the development of chemical immunoassay. However, there can be drawbacks to the use

of serum polyclonal antibodies, First, the population of antibodies in serum is dynamic with respect

87-827 CI - 88 - 7
174

Figure 2.—Monoclonal Antibody Preparation

Injection with Maintain Cell-Culture

A

Target Compound Myeloma Line

Spleen Cells

111 1

Assay for Antibody

I

Select

Desired

Clones

+ Propagate

Desired Clones

Grow m

Mass

Culture

J

Monoclonal Monoclinal

Antibodies Antibodies

SOURCE: Ralph Mumma, Pennsylvania State University and Ken Hunter, Westinghouse Bio-Analytic Systems, Co., 198B.

to concentration and quality. Secondly, the presence of an array of qualitatively different antibodies may in some cases obscure the analytical capability of the serum. Finally, the serum often bears

unwanted antibodies that bind to the carrier molecule or the spacer unit between carrier and hapten;

these antibodies, whether of natural origin or induced, may confound the analytical application.

The unwanted antibodies often can be moved by

purification steps (e.g., affinity, chromatography).

Notwithstanding these problems occasionally encountered with polyclonal antibodies, many excellent immunoassay have been developed using these

biological reagents. However, a recent technological advance now allows a single form of anti-chemical antibody to be produced in unlimited quantities.

Hybridomas and Monoclonal Antibodies

In 1975, two British scientists, George Kohler and

Cisar Milstein, discovered that somatic hybrids between B lymphocytes and myeloma cells could produce antibody of “predefined” specificity (16). That

is, the donor animal could be immunized with a target substance and immortal clones of these hybrids

that secrete one particular antibody (monoclinal)

with binding affinity for the target substance could

be isolated. The hybrid tumors are known as hybridomas, and the monoclinal antibodies secreted by

these cells have certain advantages. A generalized

schematic of the hybridoma production procedure

is shown in figure 2. Briefly, the spleen from an

appropriately immunized mouse is removed and

dissociated into a single cell suspension. These cells,

some of which produce antibody to the target substance, can be maintained in nutrient medium (tissue culture] outside the mouse, but only for a few

days. Although some specific antibody can be identified in the culture medium, it is too little to be of

practical value. However, these short-lived cells can

be physically fused in the presence of an agent such

as polyethylene glycol (10) to myeloma cells, tumors

of B lymphocyte origin that can live indefinitely in

tissue culture (28). The resulting hybrids are heterokaryons, bearing the combined genetic information or genotype of both parental cells. Of paramount importance, the hybrids express two critical

phenotypic characteristics, one derived from each

parental cell; they secrete the antibody of the parental B lymphocyte, and they have unlimited growth

potential, a trait of the parental myeloma. An elegant biochemical selection system is used to isolate

the hybridomas (18), which are subsequently cloned

to insure homogeneity. The cloned hybridomas can

be grown in mass culture where the secreted antibody accumulates in the culture medium, or they

can be adapted as ascites tumors in the peritoneal

cavities of mice where very high levels of antibody

accumulate in the ascites fluid. In either case, the

product of the cloned hybridomas is a monoclinal

antibody, a homogeneous reagent. The hybridomas

can be cryopreserved and stored indefinitely in liquid nitrogen, and the monoclinal antibody is stable indefinitely under a variety of storage conditions. Therefore, the hybridoma technology can

produce an unlimited, stable, and homogeneous

supply of monoclinal antibodies.

Principles of Chemical Immunoassays

An enormous variety of immunoassay configurations have been developed, and a thorough review

is beyond the scope of the present paper. However,
175

as all immunoassay obey the same laws of mass

action and thermodynamics, some general statements can be made. Immunoassay for small molecules such as pesticides must operate by competitive inhibition or displacement in which the binding

of the free pesticide molecule to the antibody competes or displaces a tracer molecule, By way of example we will briefly discuss enzyme immunoassay, the most widely used method that employs an

enzyme as the tracer and generates a color reaction

as the read-out. Other tracers include radioisotopes,

fluorescent molecules, magnetic particles, electron

spin labels, etc.

The enzyme immunoassay, a modification of the

original enzyme-linked immunosorbent assay of

Engvall and Perlmann (7), is conveniently performed

in a 96-well plastic microtiter plate (see figure 3),

but it can be done with tubes or test strips. Prior

to the first step in the enzyme immunoassay, the

surface of the microtiter wells is coated with an optimal concentration of target chemical-protein conjugate (figure 4). Another popular configuration

uses surface immobilized antibody, but the basic

principle of both assays is the same. The conjugate

adsorbs to the plastic surface by hydrophobic interactions, and following an incubation to assure

maximum binding, the nonadsorbed conjugate is

removed by washing with buffer containing a mild

nonionic detergent. The first addition to the coated

plate is a mixture of anti-chemical antibody and a

known concentration of target chemical. If no target chemical was added to the antibody, most of

the antibody would bind to the target chemicalprotein conjugate adsorbed to the solid surface of

the plate. The higher the concentration of target

chemical added with the antibody, the lower the

number of antibodies that bind to the solid phase

due to the competitive inhibition of their binding

sites through interaction with free target chemical

in the fluid phase. After an incubation period, the

reactants are washed away, leaving only the antibodies bound to the target chemical-protein conjugate on the plastic.

The second step of the procedure involves the

addition of a tracer to detect the surface-bound antibodies from step one. In the case of the enzyme

immunoassay, the tracer is a second antibody to

which an enzyme is attached. This second antibodyenzyme conjugate binds to the surface adsorbed

anti-chemical antibodies, and following an incubation, unbound second antibody is removed by washing, The third step in the enzyme immunoassay involves the addition of a solution of colorless enzyme

substrate, which is converted by the enzyme into

Figure 3.—Applying Reagents to 96 Well-Plate

SOURCE: Ralph Mumma, Pennsylvania State University and Ken Hunter, West

inghouse Bio-Analytic Systems, Co,, 1988.

a colored reaction product, the concentration of

which is a direct measure of the concentration of

antibody-enzyme tracer bound to the anti-chemical

antibody on the plastic surface.

Because the color reaction is directly proportional

to the number of anti-chemical antibodies bound

to the plate, it is inversely proportional to the concentration of free target chemical. The higher the

concentration of target chemical, the lower the color

reaction. By running a series of known concentrations of target chemical, one can create a standard

curve such as that shown in figure 5. A plot of optical density (color) versus target chemical concentration yields a curve with a linear portion often

extending over several orders of magnitude. The

enzyme immunoassay becomes an analytical tool

when unknown samples are run at the same time

and their optical density values compared with the

standard curve. The apparatus for analyzing the
176

Figure 4.-Competitive Inhibition Enzyme Immunoassay

b

STEP I STEP I I STEP Il l

SOURCE: Ralph Mumma, Pennsylvania State University and Ken Hunter, Westinghouse Bio-Analytic Systems, Co., 1988.

color reaction consists of a commercially available

automated spectrophotometer that can evaluate the

color in each of the 96 wells in less than one minute, a microcomputer interfaced with the spectrophotometer, and a software program for analyzing the data (see figure 6).

Enzyme immunoassay such as the one described

earlier require 2 to 4 hours to perform, and most

of this time is devoted to incubation. This assay format is highly quantitative, but other formats such

as tube and test-strip enzyme immunoassay can

be performed in minutes, and the results can be

quantitative or semiquantitative. The criteria of sensitivity, specificity, accuracy, precision, and ruggedness, all critical to any treatment of conventional

analysis, must also be addressed in chemical immunoassay. The enzyme immunoassay and similar

assays that use a standard curve for comparison and

determination of values in unknown samples are

very amenable to statistical treatment. Although

there is variation between particular chemical immunoassay due to the nature of the target chemical and the idiosyncratic properties of each antichemical antibody, chemical immunoassay can

generally be as sensitive as conventional analysis

techniques. Specificity is an inherent property of

the antibody and is defined as the spectrum of crossreactivities with related chemicals. It is not unusual,

however, to see discriminatory capability at the single atom level, or even stereochemical selectivity

(4). Accuracy and precision are more related to the

performance of the immunoassay than the properties of the antibodies, and for such assays as the

enzyme immunoassay, these criteria are comparable to most conventional analysis methods. The variation between assays and between laboratories running the same immunoassay is also comparable.

Immunoassay have component parts just as conventional assays, and these components must be

standardized. For antibodies, this means that large

batches of purified reagents must be prepared,

stored in a way that preserves their integrity, and

tested in standardized assays to ensure their quality.

Practical Applcations of Chemical

Immunoassays

Ercegovich (1976) was one of the first persons to

recognize the potential of immunoassay to pesticide residue analysis. His students and colleagues

(one of the authors, ROM) pioneered work in developing immunoassay for the organic phosphate

insecticide, parathion (1, 9, 30). Similarly, Bruce

Hammock’s laboratory was actively developing immunoassay for pesticides (32), and Ken Hunter and

colleagues (13, 14) developed antibodies to paraoxon

and other organic phosphates recognized as war

gasses. Subsequently, antibodies have been developed and reported for more than 30 pesticides, and

a number of papers have appeared reviewing this
177

Figure 5.—Standard Curve: Parathion Immunoassay

I

-3 -4 -5 -6 -7 -8 -9 -lo

LOG MOLAR CONC OF PARATHION

SOURCE Ralph Mumma, Pennsylvania State University and Ken Hunter, West.

inghouse Bio-Analytic Systems, Co , 1988

Figure 6.—Computer, Printer, and Spectrophotometer

SOURCE: Ralph Mumma, Pennsylvania State University and Ken Hunter, West.

inghouse Bio-Analytic Systems, Co., 1988.

progress (11, 12, 21). Industrial companies, some

with commercial interest in mind, have developed

many more immunoassay for pesticides, plant

growth regulators, antibiotics, and other xenobiotics, but these data have not and may never be published.

Antibodies have been developed for various classes

of pesticides, e.g., organic phosphates, carbamates,

triazines, halogenated hydrocarbons, chlorophenoxy

herbicides, pyrethroids, chitinase inhibitors, and

biorational insecticides (21). Immunoassay also

exist for a number of fungicides that cannot be

grouped easily into a chemical class, such as

Benomyl, Iprodione, maleic hydrazide, Metalaxyl,

and Triadimefon. Interestingly, the antibodies for

the pyrethroid S-bioallethrin exhibited chiral specificity (32), which cannot be achieved by any approved conventional method. An important potential use is with the biorational insecticides, such as

the exotoxins from Bacillus thuringiensis, which

can be quantified using immunological techniques.

In the future, many biological agents derived through

molecular biological techniques may be targeted for

pest management practices, and immunoassay

may be the only practical method to quantify these

organisms or agents. Two environmentally sensitive chemicals, dioxin and pentachlorophenol, can

also be analyzed in this manner, and an EPAapproved immunoassay for the latter compound is

expected shortly.

Most of the early developmental work of immunoassay has been performed in academic institutions and with polyclonal antibodies. Unfortunately, very few examples of practical applications

are documented in the literature. An exception is

that of the contribution of W.H. Newsome from the

Food Research Division, Bureau of Chemical Safety,

Health and Welfare, Canada. Newsome has developed immunoassay for several fungicides and comparatively evaluated these with conventional methods (22, 23, 25, 24, 33). Van Emon et al. (31, 1987)

have also compared immunoassay and conventional techniques in worker exposure studies with

the herbicide paraquat. The authors of this article

feel that many more application experiments need

to be performed before we can thoroughly understand the influence of the matrix on immunoassay

results.

The detectional limits of currently developed immunoassay for pesticides usually range from O. I

to 1,000 parts per billion. Pesticide tolerance limits

on many raw agricultural commodities are in the

order of parts per million, and thus, immunoassay
178

are sensitive enough to immediately make a contribution. With aqueous products such as water, fruit

juices, and milk, immunoassay can be directly performed without any cleanup steps (3, 11).

Real-Time, On-Line Agrochemlcal

Monitoring

Almost all of modern analytical chemistry deals

with discrete measurements. Unfortunately, chemicals often present dynamic problems, concentrations waxing and waning over time (e.g., groundwater contamination with pesticides). Even the

immunoassay cannot provide real-time (instantaneous), on-line (continuous) monitoring of these chemicals, However, a revolution in sensor technology

is upon us that may provide both capabilities and

may do so utilizing the same biological molecules

generated for immunoassay.

The interface of biologic molecules like antibodies with microelectronic sensor systems to create

hybrid devices known as biosensors promises to

provide analytical capabilities beyond those now

available. A biosensor is defined as a microelectronic device of one kind or another that utilizes

a biologic molecule as the sensing or signaltransducing element. The structural requirements

of a biosensor are shown in figure 7, and they include the following: a means of introducing the sample matrix to the sensor surface; an antibody or

other biological molecule with binding affinity for

a particular analyte in the matrix; a transduction

mechanism whereby the binding event generates

an electrical signal; appropriate amplification, processing, and storage of the generated signal data; and

a means of outputting the information in a usable

format.

A review of the many potential biosensors and

the principles upon which they are based is beyond

the scope of this report, and the reader is referred

to recent reviews (6, 19, 20, 15, 29).

Figure 7. —Generalized Biosensor

BIOLOGICA L

DETECTOR

MOLECULES

SOURCE: Ralph Mumma, Pennsylvania State University and Ken Hunter, Westinghouse Bio-Analytic Systems, Co., 1988.

Comparison of Immunoassay With

Conventional Methods of Pesticide

Residue Analysis

Conventional Methods versus

immunoassay Methods

Before we compare conventional analytical methods to immunological assays, we must first summarize the steps used in both procedures. In a traditional analysis the raw agricultural commodity or

processed food is first subsampled and extracted

or homogenized with an organic solvent to remove

the pesticide residue from the insoluble debris, The

organic extract is concentrated to small volume, and

then an aliquot is analyzed by gas or high performance liquid chromatography. Pesticides are usually identified by the relative time it takes them to

come through the chromatographic column (retention time) and their response to selective detectors.

Chemical specific detectors are usually used with

gas chromatography, and these detectors indicate

the presence of halogens, nitrogen, phosphorous or

sulfur atoms, which may be components of the pesticide residue. Although these detectors are highly

specific, many compounds contain these atoms, and

analyses can be confounded by such interfering

compounds. Ultraviolet detectors are often the preferred method of detection with high-pressure liquid chromatography, but these detectors are also

sensitive to all ultraviolet-absorbing substances. Because of these interference problems, organic extracts of food and raw agricultural commodities

usually have to be partially purified using organicsolvent partitioning steps and time-consuming

column chromatography prior to analysis by gas or

high pressure liquid chromatography. This partial

purification is often referred to as a cleanup step,

Conventional analytical techniques are time-consuming, require environmentally sensitive and

ultrapure organic solvents, utilize expensive chromatographic equipment, and require highly trained

technicians. This results in expensive analyses for

pesticide residues even for the most simple compounds.

However, when all procedures are followed, conventional analytical techniques are reliable, repeatable, and sensitive, Gas chromatography using

atom-specific detectors usually can detect residues

at the 1 to 100 picogram (10-9fl grams), level but

only a small amount of sample (1 to 5 microliters)

can be analyzed in this manner. With high pressure
179

liquid chromatography, a much larger aliquot of

sample can be analyzed (25 to 50 microliters), but

ultraviolet detectors usually require at least 1,000

picograms of residue, Gas chromatography separations require the chemicals to be volatile enough

for separation as a heated gas, but unfortunately,

many pesticides and their degradation products are

not volatile and cannot be identified by this method.

This is particularly true of the more polar, watersoluble pesticides and their metabolizes. Also many

pesticides do not absorb strongly in the ultraviolet

and cannot directly be quantified with high sensitivity by this technique.

Immunological assays for pesticides may also involve many of these basic procedural steps. For the

purpose of this presentation, we may think of an

antibody as a very selective detector that is particularly useful for polar and water-soluble materials.

In fact, immunological techniques are superior to

conventional techniques in the direct analysis of

pesticides in water. As for conventional methods

of analysis, immunoassay require that processed

food and raw agricultural commodities be extracted

prior to analysis. However, the cleanup steps may

be much abbreviated with the immunological technique. Also with liquid products, such as fruit

juices, milk and soups, immunological analysis can

be performed directly (3, 35). A potential drawback

to immunoassay is that they are compound-specific

and therefore most useful for single residue analysis. In contrast, conventional multiresidue procedures can detect and quantify many pesticide residues simultaneously. However, an antibody’s great

specificity does not always have to be an issue since

antibodies can be selected that detect several chemicals of related structure, and different compoundspecific antibodies can be combined in one analysis. Alternately a number of aliquots of food extract

can each be analyzed with antibodies selective for

a specific compound or for classes of compounds.

Immunoassay can be as reliable and repeatable

as conventional methods of analysis, but usually the

more highly quantitative immunoassay require

more time to run than less quantitative immunoassay, Other immunoassay configurations can be

quicker and simpler (1 to 10 minutes), but they are

usually semiquantitative. However, if the emphasis is only on pesticide levels in food that exceed

a certain concentration requiring regulatory action,

the immunoassay are superior screening techniques. Immunoassay are also readily automated,

while conventional analytical procedures are not,

Advantages and Disadvantages of

lmmunoassays

From an application standpoint, most immunoassay for pesticide residue analysis are simple and

rapid, and in some cases, they may be used without any cleanup step. They are particularly useful

for polar or water-soluble pesticides and their degradation products, and often immunoassay can easily be developed for compounds that are difficult

to analyze by conventional methods. Since regulatory laboratories do not routinely analyze for pesticides not included on their multiresidue procedures

list, immunoassay has the potential of filling this

important void.

On the negative side, we should cite that the more

rapid versions of immunoassay are usually not as

sensitive and probably not as reproducible as conventional analytical techniques. Immunoassay may

not be useful in a broad multiresidue procedure,

even though several antibodies can be combined

in one analysis. Other disadvantages include the

lack of extensive commercialization, the lack of personnel with immunoassay experience, and the lack

of knowledge and practical applications to raw and

processed food.

Constraints, Opportunities, and

Recommendations

Regulatory Agencies

Regulatory agencies at both the Federal and State

levels are too slow to adopt or encourage modern

methods and immunoassay is no exception. They

seem to be spending most of their time on validation testing rather than funding or conducting stateof-the-art analytical methods development. To cite

some examples, use of capillary column gas chromatography is only now starting to be accepted—it

has been a routine procedure in most academic laboratories for years. Solid phase extraction (SPE) or

concentration is rapidly being utilized by industry

and private laboratories but it is not being emphasized by regulatory agencies. SPE is particularly attractive, since it often eliminates the need for expensive and environmentally sensitive solvents; this

alone should be reason to encourage their use.

Immunoassay for pesticides have been demonstrated for more than 10 years, and regulatory agencies should have been taking a lead role in the development of these new techniques. Since many
180

agrochemical industries and private laboratories

have utilized these techniques for several years, regulatory agencies should encourage these organizations to make their data public so we can get a better feeling for the capabilities of these new methods.

The agrochemical industry would not be using immunoassay unless they felt they were useful and

reliable for their desired goals. Why has not the EPA

or FDA sought information on these analyses so

they could make more knowledgeable decisions?

Additionally, why do they expect all analytical

methods to meet the same strict requirements when

perhaps only a screening procedure would be sufficient? By making quantitative analyses so difficult

and expensive, regulatory agencies are actually reducing our knowledge of environmental pollutants

because present methods can analyze only a fraction of the samples that should be analyzed.

There is at least one bright light in this dim world.

The State of California has contracted for the development and testing of a number of immunoassay for pesticides. It is a pleasure to see a State

regulatory agency take the lead in developing and

evaluating this potentially important tool for the

future.

Legislative Actions

Both State and Federal legislatures have constrained the regulatory agencies because they have

asked them to do too many things and have not provided the financial backing to perform these tasks,

Legislatures could take the lead by encouraging development and applications of the new methods of

analysis. They should require the regulatory agencies to set aside a reasonable fraction of their budget moneys for developing the methods of the future. They should support grants and contracts to

companies willing to pursue developments of new

methods like immunoassay. Many new technologies such as immunoassays, enzyme assays, biosensors, solid-phase extractions, and affinity scavenging are now emerging, and much work needs to be

done to determine their capabilities. We do not envision these techniques as replacing conventional

methods, but rather as supplements to these methods. Such legislative action would stimulate development of these areas, and the well-being of the general public would greatly benefit.

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the Residue Level, Advances in Chemistry Series #104, R.F. Gould (cd.) (Washington, DC:

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al., “Development of a Radioimmunoassay for

Parathion,” J, Agric. Food Chem. 29:559-563,

1981.

Gefter, M. L., Margulies, D. H., and Scharff,

M. D., “A Simple Method for Polyethylene GlycolPromoted Hybridization of Mouse Myeloma

Cells,” Somat. Cc]] Genet. 3:231-236, 1977.

Hammock, B. D., Gee, S. J., Cheung, P. Y. K., et al.,

“Utility of Immunoassay in Pesticide Trace

Analysis,” Pesticide Science and Biotechnology,

R. Greenhalgh and T.R. Roberts (eds.) (Oxford,

G. B.: Blackwell Scientific Publications, 1987).

Hammock, B.D. and Mumma, R. O., “Potential

of Immune-Chemical Technology for Pesticide

Analysis,” Pesticide Analytical Methodology,

Am. Chem. Soc. Symposium Series No. 136, pp.

321-352, J. Harvey, Jr., Zeig (eds.) (Washington,

DC: Am. Chem. Soc., 1980).

Hunter, K.W. and Lenz, D. E., “Detection and

Quantification of the Organophosphate Insecticide Paraoxon by Competitive Inhibition Enzyme Immunoassay,” Life Sci. 30:355-361, 1982.

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633-636, 1987.

Kohler, G. and Milstein, C., “Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity,” IVature 256:495-497, 1975.

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and Biotechnology, R. Greenhalgh and T.R.

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182

Federal Pesticide Monitoring Programs:

Analytical Methods Development

Charles L. Trichilo and Richard D. Schmitt, U.S. Environmental Protection Agency, Office

of Pesticide Programs, Hazard Evaluation Division, Residue Chemistry Branch, Washington, DC

Conents

Page

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ...182

Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..183

Tolerances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....183

Qualitative Data on Metabolism and Degradation. . ............................183

Metabolism in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................183

Metabolism in Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................183

Quantitative Data on Magnitude of the Residue . . . . . . . . . . . ....................184

Magnitude of the Residue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................184

Processing Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......................184

Feeding Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................185

Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......................185

Effect of Limited Tolerance Data on Analytical Methods . ....................185

Importance of Analytical Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....186

Summary of Key Inputs to Food Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .186

Overview Of FDA, USDA, and EPA Needs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...186

FDA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................186

USDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..............................187

EPA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....187

Analytical Methods Development Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......188

FDA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .................188

USDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..............................188

EPA, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..................,..188

Sharing Information Among Agencies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........189

Accessing Technology in the Private Sector . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ....190

Dealing With Hazardous Pesticides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ....190

Areas For Improvement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....................190

Recommendations For Improving Methods Development Programs . .............190

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .........................190

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .190

Figure 8

Figure Page

l. Residue Chemistry Data Requirements. . . . . . . . . . . . . . . . . . . . . . ...............184

2. Key Input to Food Monitoring. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .187

This paper provides a brief overview of the needs

of the U.S. Environmental Protection Agency (EPA),

Food and Drug Administration (FDA) and United

States Department of Agricuhure (USDA) in the

area of analytical methods for monitoring pesticide

residues in food. The importance in developing effective methods for tolerance enforcement that are

rapid, sensitive, and inexpensive is discussed. The

congressional mandates and agency approaches related to food residue monitoring, tolerance enforcemerit, and methods development are also described.

The effects of (l) changes in agricultural use practices that increase the extent of pesticide residues

in the food supply and (2) limited tolerance data on

the methods development process are noted. The

acquisition of adequate metabolism data is the single most important chemistry contribution to the

methods development process. Without full knowledge of the chemical identity of significant metabo-
183

lites that occur as residues in food, it is impossible

to develop monitoring methods for all residues of

concern. While the primary focus for tolerance enforcement is on analytical methods, of equal importance is the need for readily available analytical

reference standards.

The contribution analytical methods have in providing monitoring feedback for tolerance enforcement and tolerance-setting, in addition to their role

in reducing the uncertainty in risk assessment, is

also noted. Finally, suggestions are made for improving analytical methods for monitoring pesticide

residues in food,

Background

Prior to any discussion on analytical methods to

improve the monitoring and enforcement of tolerances for pesticide residues in the food supply, a

brief overview of tolerances and related terminology is needed. Since tolerances depend on the state

of scientific and technical knowledge (including

analytical methods) at the time they are established,

any limits in the existing data used will impose a

corresponding limit in the analytical method used.

Without an understanding of the key data elements

that lead to a tolerance, it will be difficult, if not

impossible, to significantly improve the analytical

methods or the method development process for

better tolerance enforcement (l).

Tolerances

A tolerance is the legal maximum residue concentration of a pesticide chemical allowed in a food

or feed. Tolerances minimize uncertainty about

food safety with regard to those pesticide residues.

If a pesticide is detected and residues exceed the

tolerance or no tolerance is established, the crop

may be considered adulterated and be seized by the

Food and Drug Administration (FDA), the United

States Department of Agriculture (USDA), or a State

enforcement agency. EPA establishes tolerances for

pesticides, while FDA, USDA, and the States carry

out tolerance enforcement in foodstuffs. EPA also

provides the analytical standards used intolerance

enforcement (2).

Tolerances are set under authority of the Federal

Food, Drug, and Cosmetic Act (FFDCA). Section

408 of the FFDCA applies to residues on raw agricultural commodities (RACs) and Section 409 applies to processed food or feed. Section 409 includes

the Delaney Clause, which specifically prohibits the

use of cancer-causing agents as food or feed additives.

There are three types of residue chemistry data

that are essential for establishing tolerances:

1. Qualitative Data on Metabolism and Degradation

2. Quantitative Data on Magnitude of the Residue

3. Analytical Methods

The purpose of these chemistry data is to answer

two basic questions. First, what is the chemical residue? Second, how much residue is there? Analytical methods are essential in providing answers to

these two fundamental exposure questions. The

“what” and “how much” information is used by

EPA toxicologists to determine whether the dietary

exposure is acceptable. The first half of EPA’s

tolerance-setting job is completed when EPA has

concluded what and how much residue is present

and that this level of residue is safe. The other half

of EPA’s job is to be sure adequate enforcement

methods are available to check that the residue

levels in the food supply do not exceed the tolerances.

@qualitative Data on Metabolism

and Degradation

In order to answer the “what is the residue” question, qualitative data are required to determine the

identity of the pesticide residues resulting from the

transformation in plants and animals. EPA refers

to these transformation studies that include both

pesticide degradation and metabolism as metabolism studies.

Metabolism in Plants

Plant metabolism data characterize the identity

of the residue that occurs in crops intended for

consumption as a food or animal feed, These data

identify the pesticide residues that remain in agricultural crops as the result of environmental transformation processes (degradation and metabolism).

The resulting residue at harvest may be different

than the chemical applied, due to breakdown or metabolism of the applied pesticide.

Metabolism in Animals

Whenever use of a pesticide results in residues

in a livestock feed, or when a pesticide is applied

directly to livestock, animal metabolism studies are

required. The resulting data identify the pesticide

residues to look for in the edible tissues of livestock

or milk and eggs that result from transformation

processes in the animal. If feed items are not involved or if this exposure pathway is blocked by

label restriction, these data are not required (see figure 1).
184

Figure 1 .—Residue Chemistry Data Requirements

Residue data Residue data

NOT required required

All residue Is the PLANTS

data waived, No No pesticide Yes Yes Metabolism

no method to < Used on > Methods

check misuse Food crops? Magnitude

Meat & milk Detectable

Data No No Residues

Processing < in the

Data Crop?

Meat & milk

Data Waived No Is the crop Yes

-

No meat & No used as Yes -

milk methods < livestock feed? > Methods

I J

ANIMALS

Metabolism

Feeding Studies

I

Processing Is the Processing

Studies Waived No

\ No Crop Yes Yes Studies

No processed

Commodity Methods e Processed? > Methods for Processed Commodity

4 I 1

Food Additive No Do Yes Establish

Tolerances No Residues Yes Food Additive

Not Needed ~ Concentrate? ❑ Tolerance

I 1

SOURCE: Charles Tnchllo and Richard Schmitt, Envmcmmental Protection Agency, 198S

Quantitative Data on Magnitude of

the Residue

Magnitude of the Residue

After the metabolism data have indicated what

residue to look for in plants and animals (if applicable), methods are developed to measure these

residues, Actual residue field trials are carried out

to quantify the residues. These are the studies in

which the pesticide is applied to crops at known

application rates, in a manner similar to the use

directions that will eventually appear on the label.

The residue field trial studies result in residue data

for the raw agricultural commodity (RAC) as it

travels in interstate commerce.

Processing Studios

Processing studies are required to determine

whether residues in raw commodities can concentrate or degrade on processing. If residues concentrate on processing, food or feed additive tolerances

must be established. If residues do not concentrate

on processing, the tolerance on the RAC applies to

all processed food or feed derived from the RAC,

It should be noted that the current EPA legal opinion on the Delaney Clause is that it applies to food
185

and feed additive tolerances but not to RAC tolerances.

feeding Studies

Livestock feeding studies are required whenever

residues result in or on crops that are used as feed

items. These studies provide data on the quantitative transfer of residues to meat, milk, poultry, and

eggs. These studies are also required if a pesticide

is applied directly to livestock.

Analytical Methods

Analytical methods serve two important purposes. The first is to generate residue data on which

the dietary exposure assessment is based. The second is to enforce the tolerance after it is established.

It should be noted that plant and animal metabolism data are the most critical data that precede the

development of analytical methods. Without proper

and complete metabolism studies to indicate which

residues to look for, the development of analytical

methods for all residues of concern may not be possible (3, 4).

Since risk assessment depends on exposure, analytical methods can have a significant impact on

reducing the uncertainty in risk assessments by providing needed exposure data.

Analytical methods also serve an important role

in providing feedback for tolerance enforcement

and tolerance-setting procedures. Routine monitoring provides the obvious feedback on whether tolerances are being exceeded, or on whether tolerances have been set too high. However, sometimes

the results of tolerance enforcement can lead to

needed changes in the tolerance-setting process. For

example, FDA monitoring uncovered over-tolerance

residues of malathion in grain dust. Grain dust is

now routinely collected at grain storage sites to prevent dust explosions and has become a disposal

problem. Recently, the industry began pelleting this

dust and using it as an animal feed. Due to the high

concentration of pesticide residues in the dust, feeding of grain dust could lead to detectable residues

in meat or milk. Furthermore, feeds formulated with

grain dust as an ingredient are subject to seizure

by FDA. As a direct result of the monitoring by FDA,

EPA established a 135 ppm tolerance for malathion

on grain dust (5). EPA also is revising its tolerancesetting procedures to routinely establish tolerances

for grain dust to ensure that any potential residues

in meat and milk are covered by tolerances and are

safe.

Effect of Limited Tolerance Data on

AnaIyticaI Methods

Any limits in the data that are used to establish

tolerances will have a profound impact on the analytical methods. From EPA’s perspective, there are

two areas that can have a significant effect on the

current state of the adequacy of analytical methods:

1) incomplete metabolism data, and 2) missing or

impractical label restrictions that do not block exposure pathways,

Analytical methods can only be developed for

those components of the residue that are identified

in the metabolism studies. If metabolism studies do

not fully identify the residues present, important

components of the residue may remain undetected.

Older chemicals whose metabolism studies fail to

identify the significant residues present constitute

the largest problem here. From EPA’s experience

in reviewing older chemicals as part of the re-registration process, it is not uncommon for 50 to 80

percent of the 14C residues in radiolabeled metabolism studies to be unidentified. These limited data

have an important effect on the ability to develop

analytical methods, The development of analytical

methods for chemicals with significant metabolism

deficiencies will be delayed until the needed residue identification work is completed.

It should be noted that the complete set of residue data are not always required, particularly if the

exposure pathways that lead to residues moving further into the food chain can be blocked by practical

label restrictions. Determining what is practical is

subject to much judgment and is further complicated by the dynamics of changing customary agricultural practices; this includes both economic and

weather conditions that may affect the supply and

demand of food or feed items.

In general, label restrictions are considered practical if three criteria are met: 1) the crop is under

the direct control of the grower; 2) the economic

value of the crop as a feed item is low; and 3) the

U.S. customary practice is not to use the item as

a feed. For example, label restrictions against feeding corn forage to prevent residues from moving

into meat and milk commodities are not practical.

Even though corn forage is under direct grower control, the high value of the feed item and the overwhelmingly common practice of feeding this commodity makes the restriction impractical.

What was practical at a certain period of time can

change as use practices change. For example, until

recently EPA considered the feeding directive, “Do

not feed sugar beet tops” to be a practical restric-
186

tion. Accordingly, data on metabolism, magnitude

of the residue, and analytical methods were waived,

since the feeding restriction was expected to prevent residues from moving into meat and milk.

Therefore, analytical methods for determining residues in meat and milk were not available, since no

pesticide residues were expected in these environmental media. In recent years, sugar beet tops have

increased in economic value so much that California growers have changed their customary practice

and now sell the beet tops for livestock feed. In this

case, EPA was aware of the change and required

data, including analytical methods, to cover any

residues that could be expected in meat and milk.

However, EPA is not always aware of changes

in use practices that result in residues moving further into the food chain than originally expected.

The EPA Re-registration/Registration Standard

process is one systematic scheme to identify such

a problem area and call in the needed data. Again,

however, until all chemicals are given a current review, the potential will exist for changes to occur

in use patterns that result in more residue in the

food supply with no corresponding analytical methods for enforcement.

It is important to note that no residue data are

required for all nonfood uses. For nonfood uses,

analytical methods are not required for detecting

pesticide residues in food or feed crops, since residues are not expected in the food chain. Some older

uses, previously considered as nonfood uses, may

now actually be food uses that require residue data.

Until these situations are identified, monitoring for

food residues may not be possible because analytical methods are lacking. For these previously classified nonfood-use chemicals, analytical methods

may not be available to FDA, USDA, and the States

to check for accidental contamination or illegal use

in food and feed.

Importance of Analytical Standards

Up to this point, the importance of analytical

methods for monitoring pesticide residues in foods

has been the primary emphasis of the Office of Technology Assessment workshop. Of equal importance,

however, are the analytical standards that are used

in the laboratory by those chemists conducting monitoring or enforcement activities. An analytical

standard is a high purity reference standard used

to calibrate the detector response of an analytical

method. Chemists use a known analytical method

together with a known analytical standard whose

behavior and response is very predictable under laboratory conditions. Use of inappropriate standards

or standards of low purity will lead to erroneous

methods results, If analytical standards are not of

sufficient purity, enforcement becomes more timeconsuming and difficult as predictable behavior

cannot be obtained. If analytical standards are not

available, enforcement becomes difficult, if not impossible.

When EPA is aware that analytical standards are

not available, the agency can act under its authority under FIFRA 3(c)2(b) to require the pesticide

registrant to submit additional quantities of the analytical standards. Failure of a registrant to provide

or maintain analytical standards in the EPA repository can result in cancellation of the U.S. registration, EPA cooperates with FDA by providing analytical standards for those pesticides not having U.S.

registrations that FDA needs to monitor imports.

It should be noted that for pesticides used on imported foodstuffs that are not registered in the

United States, there is no similar mechanism to obtain standards if the foreign registrant does not wish

to cooperate.

Summary of Key lnputs to Food

Monitoring

The importance of(1) changes in agricultural use

practices that increase the extent of pesticide residues in the food supply, (2) limited tolerance data

on residue identification (metabolism studies), and

(3) the availability of analytical reference standards

to food monitoring methods is depicted in figure

2. Without full knowledge of the chemical identity

of significant metabolizes that occur as residues in

food, it is impossible to develop monitoring methods for all residues of concern. Similarly, the absence of analytical standards or the lack of knowledge about any increase in the extent of pesticide

residue involvement of the food chain due to changes

in agricultural use practice severely hampers the

methods development process.

Overview of FDA, USDA, and EPA

Needs

FDA

The Food and Drug Administration’s (FDA) congressional mandate for enforcing tolerances is contained in the Federal Food, Drug and Cosmetic Act

(FFDCA). The FDA is responsible for monitoring

and enforcing tolerances for pesticide residues in

all foods and feeds except meat and poultry. They

need rapid, inexpensive methods for a wide variety of food matrices. FDA relies primarily on mul-
187

Figure 2.— Key Input to Food Monitoring

I FOOD MONITORING I

I I

I

I I

I I I I

IANALYTICAL METHoDsl ANALYTICAL STANDARDS

CURRENT USE INFORMATION

SOURCE: Charles Tnchilo and Richard Schmitt, Environmental Protection

Agency, 1988

tiresidue methods that they have developed to handle the bulk of their monitoring efforts. The driving

force for FDA to develop these methods is the great

economic savings associated with methods capable of determining many pesticides at one time, The

five multiresidue methods used by FDA detect approximately 60 percent of the pesticides with tolerances in food. FDA also uses single-chemical methods for monitoring pesticides of special concern

when these pesticides are not detected by the multiresidue methods. The FDA also carries out marketbasket surveys to determine the level of residues

of many pesticides in ready-to-eat food. The FDA

compiles the results of these quantitative analyses

for pesticide residues in food, Data including incidence and levels of pesticide residues are available

to EPA, the World Health Organization (WHO), and

other interested parties (l). FDA also publishes their

enforcement methods in the Pesticide Analytical

Manual (PAM), Volume I (PAM I) (6) of this manual includes sampling procedures and a description

of the multiresidue analytical methods. Volume II

(PAM II) (8) of this manual includes methods for

detecting individual pesticides.

The U.S. Department of Agriculture’s (USDA)

congressional mandate for enforcing meat and poultry tolerances is included in the Meat Inspection

Act and the Poultry Inspection Act. The USDA enforces pesticide tolerances for meat and poultry.

USDA is also responsible for enforcing drug residue tolerances established by FDA. They also need

rapid, inexpensive methods for meat and poultry

products, (These include meat and poultry muscle,

tissue, fat, liver, kidney, and processed meat products.) While USDA relies on multiresidue methods

for chlorinated hydrocarbons (9), they also use individual methods for specific pesticides and animal drugs in their enforcement programs. These

methods used by USDA are also developed by

USDA. Recently, USDA has begun developing and

utilizing rapid screening methods for specific compounds so that the more expensive laboratory methods will only be used on samples likely to be contaminated.

EPA

EPA’s congressional mandates come from the

FFDCA and the Federal Insecticide, Fungicide and

Rodenticide Act (FIFRA). EPA sets tolerances under the FFDCA and registers pesticides under the

FIFRA. EPA is responsible for establishing pesticide tolerances for all foods and feeds. Although

EPA has no direct tolerance enforcement responsibility, the agency shares in the need for practical

methods that are readily available. Practical methods need to be rapid, inexpensive, and reproducible, and they must involve equipment and reagents

that are commercially available (10).

EPA does not normally develop analytical methods for tolerances. Rather, EPA requires the registrant of the pesticide chemical to develop methods

necessary to enforce tolerances (11), EPA has provided written guidelines for the details on how this

work should be carried out (12, 13, 14). EPA laboratories carry out method trials to assure that these

written methods can actually be used to enforce tolerances.

To facilitate food monitoring and tolerance enforcement activities, EPA includes a methods availability statement in each Federal Register (FR) tolerance notice so that Federal and State enforcement

agencies and other interested parties can more readily obtain copies of the methods. EPA also sends

copies of methods for enforcing tolerances to FDA

for publication in Volume II of the Pesticide Analytical Manual (7). In cases where no methodology

exists for a pesticide of concern, EPA has taken the

lead and developed methods such as was done to

quantify dietary exposure to unsymmetrical dimethyl

hydrazine (UDMH), a degradation product of daminozide (15). EPA’s goal is to assure that a method suitable for enforcing tolerances is available before a

pesticide tolerance is established as well as for all

existing tolerances.
188

EPA accepts single chemical methods as being

suitable for enforcement. In 1984, EPA regulations

were revised to require data on whether existing

FDA and USDA multiresidue methodology will detect and identify the pesticides (16).

To improve the quality of single chemical methods, EPA has encouraged more collaboration by

method users, and it has encouraged petitioners to

conduct an independent method check by a second

laboratory prior to submitting the tolerance enforcement method to EPA. More recently, EPA (17, 18)

has formally proposed independent laboratory confirmation for tolerance methods (19).

Analytical Methods Development

Program

FDA

The FDA’s primary method development efforts

are in the area of multiresidue analytical methods

(20). If multiresidue methods are impractical or impossible, single residue methods are developed to

insure that residues can be determined. FDA uses

information in the Surveillance Index to arrange

in order of importance the methods development

for pesticides used domestically. FDA has ranked

pesticides according to the importance of generating monitoring data. This ranking scheme is called

the Surveillance Index. The FDA Surveillance Index for pesticides was developed as a result of a

recommendation of an FDA study group (21). The

study group felt that selection of chemicals for monitoring should be based on potential health risk

rather than analytical method availability.

FDA also uses a data base (22) on pesticide use

in foreign countries to identify pesticides used outside the United States, for which methods must be

developed. FDA has five major goals in the area of

analytical methods development for pesticide residues:

Expansion of Existing Multiresidue Analytical

Methods to Additional Pesticides and Alteration

Products. Five multiresidue methods are regularly

used by FDA, and each is undergoing study for expansion to additional chemicals. Multiresidue methods for groups of certain pesticides (e.g., triazine

herbicides, chlorophenoxy herbicides, fumigants)

are also available and used on occasion.

Extension of Methods to Different Food or Feed

Commodities. This is a continuing activity dealing

primarily with multiresidue methods. Modifications to existing methods are often required before

the method can be used on additional commodities

due to different physical or chemical composition

or limits of detection.

Validation of Analytical Methods. It is the general practice to conduct a limited interlaboratory

trial among a few FDA laboratories of a new method

prior to introducing it for field use. The ultimate

goal is collaborative study of a regularly used method

for AOAC acceptance as an official method,

Adaptation of Newly Available Analytical Techniques for Integration into Existing Methods. Advances in instrumentation and sample preparation

have the potential to allow for modification of existing methods so that the methods become cheaper

and faster. FDA currently has programs on high performance liquid chromatography (HPLC), capillary

column gas chromatography (GC), computer-assisted

instrumentation, and a new residue extractor for

fatty foods.

Development of “New” Analytical Methods or

Techniques. This includes the development of immunoassay residue method capabilities (materials

for use in FDA monitoring not now commercially

available) being undertaken via contract.

In general, the methods development research

could be divided into two broad types: 1) that which

deals with the immediate program needs, and 2) that

which is directed to future goals of greater scope

to solve particular problems or to improve overall

effectiveness or efficiency. Most of FDA’s effort is

forced into the first type.

USDA

Development of residue analytical methods by

USDA comes under the purview of the Food Safety

and Inspection Service (FSIS). The FSIS method development program is currently emphasizing the

development of multiresidue screening methods,

many of which are based on immunoassay techniques. Secondary emphasis is being placed on conventional chemical qualitative/confirmatory procedures. Methods are developed both in-house and

under contract. USDA finds the meat and poultry

methods developed by pesticide producers (PAM

II methods) to be too long and expensive to be practical in a large-scale monitoring program. All methods used by USDA are subjected to collaborative

studies prior to being used in FSIS laboratories.

USDA does in-house collaborative studies and cooperates with the Association of Official Analytical Chemists (AOAC) in carrying out collaborative

studies.

EPA

Since EPA has no direct responsibility for enforcing tolerances, methods development for residues

in food is not generally carried out in EPA labora-
189

tories. Methods development at EPA is carried out

primarily by the Office of Research and Development (OR D). ORD does not have a specific program

to develop methods to detect pesticide residues in

food; however, it supports such efforts by providing analytical reference standards and technical information through its Pesticides and Industrial

Chemicals Repository (2). As noted previously, the

availability of analytical standards areas important

as analytical methods in tolerance enforcement.

ORD does develop methods to meet specific congressional mandates under a number of laws:

Clean Air Act (CAA)

Clean Water Act (CWA)

Safe Drinking Water Act (SDWA)

Resource Conservation and Recovery Act (RCRA)

Comprehensive Environmental Response, Compensation and Liability Act (CERCLA, Superfund)

Toxic Substances Control Act (TSCA).

In some cases, these methods can be used as a

starting point for detecting residues in food. The

EPA organizations responsible for administering

or implementing specific environmental laws request ORD to develop methods through research

committees consisting of ORD and program office

representatives. ORD laboratories are then directed

to perform the requested work, which they perform

internally or by contract, grant, or cooperative

agreement. Most analytical method development

activities are conducted by the Office of Acid Deposition, Environmental Monitoring and Quality Assurance (OADEMQA) in ORD.

As pesticides become of concern to the program

offices, they are sometimes included in multiresidue

methods. For example, six multiresidue methods

have been developed by the Environmental Monitoring and Support Lab (EMSL) to detect approximately 120 pesticides and degradation products in

ground water. Pesticide methods are also developed

to monitor pesticide residues for specific projects

requested by the Office of Pesticide Programs. In

most cases, existing methods available from the

literature, the FDA, or a pesticide manufacturer are

modified for the matrix of interest.

The Office of Pesticide Programs has laboratories

at Beltsville, MD, and Bay St. Louis, MS, that are

primarily responsible for carrying out method validations to assure adequate methods are available

to enforce tolerances. These laboratories have, on

occasion, developed single chemical methods for

pesticides or their metabolizes when existing methodology was unavailable for important chemicals

of concern, such as those chemicals involved in the

EPA Special Review Process.

Sharing Information Among

Agencies

Information on analytical methods is shared

through implementation of Memorandums of Understanding (23) and periodic meetings. Currently

EPA, FDA, and USDA meet quarterly to discuss specific problems associated with analytical methods.

Past results from these meetings have included the

following:

Protocols to be used by the pesticide registrants

to determine whether pesticides would be detected

by FDA multiresidue methodology (24).

User response sheets included in PAM II so that

problem methods can be identified and better methodology required of registrants (25).

Identification and prioritization of problem methods so that better methodology can be developed.

Current projects under consideration at these

meetings include the development of a protocol for

determining the acceptability of a method and writing specific criteria on the acceptability of methods for enforcement purposes.

FDA and EPA cooperate on the Surveillance Index project. EPA provides FDA with pertinent exposure and toxicology information for those pesticides with tolerances so that FDA can rank the

pesticides in order of priority for monitoring. Similarly, EPA representatives sit in on USDA’s surveillance advisory team meeting to provide input

on priority pesticides to be monitored in meat and

poultry.

EPA has recently initiated procedures to make

analytical methods submitted by pesticide tolerance

petitioners more readily available to FDA, USDA,

the States, and other interested parties. EPA now

sends FDA and USDA copies of new chemical

methods and method modifications for use on additional crops upon receipt of these methods. This

provides the enforcement agencies an opportunity

to comment on the suitability of these methods early

in the tolerance-setting process and prior to approval of the tolerance. EPA also includes, in each

published Federal Register notice for every tolerance, a specific statement on the availability of the

analytical methodology. If the method has not yet

been published in the PAM II, the FR notice includes the address of the EPA/FOI Office from

which the method can be obtained.

87-827 0 - 88 - 8
Accessing Technology in the Private

Sector

EPA requires registrants of pesticide chemicals

to submit analytical methods as part of the data necessary to register a pesticide. These methods, developed by the agrochemical producers, are made

available by publication in PAM II by FDA and released by EPA under the FOI Office. The publication of environmental matrix methods is done by

the National Technical Information Service (NTIS).

Since these methods must be made available to enforcement agencies and interested parties, EPA no

longer accepts methods that are claimed to be Confidential Business Information (CBI).

Much residue data is generated by the food processing and distribution industry. EPA is currently

working with the National Food Processors and the

Grocery Manufacturers of America to make these

residue data available for use by Federal agencies.

EPA/FDA/USDA chemists are also meeting with

technical committees of these organizations to suggest areas for monitoring pesticide residues and

new methods development.

Dealing with Hazardous Pesticides

EPA evaluates potentially hazardous pesticides

that appear to meet or exceed certain risk criteria

through its Special Review process. Under the Special Review process, all available data on toxicity

and exposure are reviewed. In addition, data essential to the determination of risk of a pesticide are

required from the registrants when appropriate.

When necessary for Special Review decisions,

EPA requests special monitoring programs from

FDA and USDA to determine the level of residues

in food. EPA also notifies the Grocery Manufacturers of America (GMA) and the National Food

Processors of America (NFPA) so that residue data

from the food industry can be made available. Increased cooperation in this area will improve the

government’s ability to deal with hazardous pesticides.

of these changes were initiated for the sole purpose

of improving the capability of enforcement agencies to monitor for pesticide residues in food.

Recommendations for Improving

Methods DeveIopment Programs

The major need among Federal and State agencies in the area of pesticide food monitoring is the

development of quicker, more comprehensive multiresidue programs. The following are suggestions

for improving methods development for food monitoring:

●

●

●

Closer coordination between EPA, FDA, and

USDA in methods research and prioritization.

Agencies should identify lead organizations for

each area of methods research and attempt to

minimize overlap.

Congress should consider providing incentives

to industry, academia, and the States to develop

methods for pesticide residues in food and to

monitor for pesticides in food.

The pesticide producers and the food Production “industry should increase their efforts at

monitoring for pesticide residues in food and

should share monitoring results with Federal

agencies.

Acknowledgments

The authors express their appreciation to Paul

Corneliussen for providing information of FDA’s

activities and to Bart Suhre for assisting with the

section on USDA’s activities. The authors also appreciate the diligence shown by Fannie Mosley in

typing and retyping this manuscript particularly under the short timeframe.

1,

2.

Areas for Improvement

Each agency should review its current regulations

and guidelines with the goal of improving or modifying them if needed, so that analytical methodology needs can be better addressed. The EPA has

issued regulation modifications involving multiresidue method protocols (27) and is considering

second-lab validation of enforcement methods. Both

3.

4.

References

Trichilo, C. L,, “EPA Pesticide Contaminant

Concern for Residues in Food and Feed,” Cereal

Foods World 32 (11):806, November 1987.

Analytical Standards are available from the US.

EPA Environmental Research Center, Environmental Monitoring Systems Laboratory, Quality Assurance Division, Las Vegas, NV.

Kovacs, M. F., “Regulatory Aspects of Bound

Residues, ” (Chemistry) Residue Reviews 97:1-

17, 1986.

U.S. EPA, Pesticide Assessment Guidelines,

Subdivision O, Residue Chemistry, Series 171-

4 (a) (l)&(2), “Nature of the Residue: Plants,” Addendum on Date Reporting (1987) Environmental
191

5.

6.

7.

8.

9.

10,

11.

12.

13.

14.

15.

16.

17.

Protection Agency, Washington, DC. (Available

from National Technical Information Service,

5285 Port Royal Rd., Springfield, VA 22161.

DOC. No. PB 87-208641).

21 CFR 56.270, April 15, 1987.

U.S. Department of Health and Human Services, Food and Drug Administration, FDA/EPA,

Pesticide Analytical Manual, Volume I [Methods Which Detect Multiple Residues).

Read, et al., The FDA Pesticide Monitoring Program, JOACAC 591-595 (1987).

U.S. Department of Health and Human Services, Food and Drug Administration, Pesticide

Analytical Manual, Volume II, Methods for Individual Residues,

Official Methods of Analysis of the Association

of Official Analytical Chemists, S. Williams (cd.)

14th Ed,, 1984, Section 29.037.

Trichilo, C. L., “The Challenge for Analytical

Chemistry, Where Methods Came From,” Food

and Drug Law Institute Conference (Sept. 11,

1987) on “Pesticides in Foods Dealing with the

Problem, ” page 2,

40 CFR 158.125 (5)(4).

Pesticide Assessment Guidelines, Subdivision

O, Residue Chemistry Series 171-4 (b), 1982, U.S.

Environmental Protection Agency, Washington,

DC. (Available from National Technical Information Service, 5285 Port Royal Rd., Springfield, VA 22161. Dec. No. PB 83-153981.)

U.S. EPA, Hazard Evaluation Division, Standard Evaluation Procedure, Analytical Methods,

Draft of 1/19/88.

U.S. EPA, Data Reporting Guidelines - Addenda

to the Pesticide Assessment Guidelines, Subdivision O, Addendum 2, Magnitude of the Residue: Crop Field Trials, Analytical Method(s),

and Storage Stability Study; National Technical Information Service, PB 86-248192.

Wright, D., Jr., “New Method for the Determination of 1,1-Dimethylhydrazine Residues in

Apples and Peaches,” J. Assoc. Off Anal. Chem.

70(4):718, 1987.

40 CFR 158.125 (b)(15).

Trichilo, C. L., “Tolerance Setting procedures

for Residues in the Food Supply,” National Agricultural Chemicals Spring Conference, 1987,

page 9.

18.

19.

20.

21

22.

23,

24.

25.

26.

27.

Kovacs, M. F., Jr. and Trichilo, C. L., “Regulatory Perspective of pesticide Analytical Enforcement Methology in the United States, ” J. Assoc.

Off Anal. Chem. 70(6):940, 1987.

EPA Office of Pesticide Programs “Notice to

Manufacturers, Formulators & Registrants of

Pesticide Products” Draft PI? IVotice 88-4, Tolerance Method Trials—Independent Laboratory

Confirmation April 1988.

McMahan, B.M, and Burke, J. A., “Expanding

and Tracking the Capabilities of Pesticide Multiresidue Methodology Used in the Food and

Drug Administrations’s Pesticide Monitoring

Programs,” J Assoc. Off Anal. Chem. 70(6):

1072, 1987.

FDA Monitoring Programs for Pesticides and

Industrial Chemical Residues in Food, Study

Group on FDA Residue Programs, June, 1979,

HEW Publication No. (FDA) 79-2116.

World Agrochemical Data Bank, an on-line service available only to subscribers to Battelle’s

World Pesticides Programme; Battelle Memorial

Institute, Research Management & Economic

Analysis Dept., Columbus, OH, 42301.

Memorandum of Understanding among the

Food Safety and Inspection Service and Agricultural Marketing Service, U.S. Department of

Agriculture and the Food and Drug Administration, U.S. Department of Health and Human

Services, and the U.S. Environmental Protection Agency, October 5, 1984, 12 pages.

U.S. Department of Health and Human Services, Food and Drug Administration, Appendix

II, Pesticide Analytical Manual, Volume I.

U.S. Department of Health and Human Services, Food and Drug Administration, Method

Evaluation Form, November, 1985, Pesticide

Analytical Manual, Volume 11.

Methods are available from the Pesticide Analytical Manual, Volume 11 or Information Service Section, Program Management and Support

Division (TS-757C), Office of Pesticide Programs,

U.S. Environmental Protection Agency, 401 M

Street, S.W., Washington, DC 20460.

Federal Register, Volume 51, No, 187, Friday,

September 26, 1986.
192

Pesticide Analytical Methods Development

at the State Level

William Cusick, California Department of Food and Agriculture, Division of Inspection

Services, Chemistry Laboratory Services

and

James W. Wells, California Department of Food and Agriculture, Division of Pest Management, Pesticide Enforcement Branch

Page

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , .. .192

Introduction . . . . . . . . . . , . . . . . . . . ... , . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . ., , .. .192

Pesticide Residue Monitoring Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..,192

U.S. Department of Health and Human Services, Food and

Drug Administration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . ...193

Florida Department of Agriculture and Consumer Services (FDACS) , .....,....193

Massachusetts Department of Public Health (MDPH) . ................,,....,.194

Montana Department of Food and Agriculture (MDA) . . . . . . . . . . . .........,...194

California Department of Food and Agriculture (CDFA) . ...................,.194

Analytical Methods Needed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .........,.196

Analytical Methods Developed at State Level . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .,.I97

Impact of Local Regulatory Initiatives . . . . . . . . . . . . . . . . . . . . . . . ................199

Role of States in Analytical Methods Development . ..............,............199

References, . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....200

Abstract

The role of state regulatory agencies in the enforcement of pesticides residue tolerances and the

development of new analytical technologies are determined by Federal policy and State legislative intent. State programs are focused to complement the

regulatory activity of the various Federal agencies,

but also to meet the state’s sometimes more stringent regulatory requirements. This paper discusses

the states’ role in the national food protection program and highlights the differences between the

state and Federal programs.

lntroduction

The U.S. Department of Health and Human Services, Food and Drug Administration (USFDA) is the

Federal agency responsible for monitoring pesticide

residues in food. Over the years, a number of states

have developed their own pesticide residue monitoring programs in response to specific needs identified by the states, perceived limitations in the

Federal program, and perhaps most important, in

response to the increased consumer concern regarding toxics in the food supply. The size and goals

of these state programs vary, depending on the perceived need in the state and the funding available.

This paper will examine the pesticide residue monitoring programs of several selected states in comparison with that of USFDA; discuss the kinds of

analytical methods most needed, including a discussion of the applicability and potential for emerging analytical techniques; provide an overview of

present analytical methods development in the

states; discuss local regulatory initiatives that have

placed special analytical requirements on the state

laboratories; identify the present role of the state

in analytical methods research and development;

and make suggestions for what that role may be in

the future.

Pesticide Residue Monitoring

Programs

In order to present information that would be

more representative of the national status of states’

pesticide residue monitoring programs, examples

are provided from several states with programs of
193

different sizes and objectives. The states included

are Florida, Montana, Massachusetts, and California.

U.S. Department of Health and Human

Services, Food and Drug

Administration

The U.S. Department of Health and Human Services, Food and Drug Administration (USFDA) analyzes approximately 10,000 samples of fresh fruits,

nuts, and vegetables each year (7). Samples are taken

of imported produce, as well as domestic produce

destined for interstate shipment. The USFDA program consists of two major components: compliance monitoring and surveillance monitoring. In

this paper we will deal with the surveillance monitoring component because it has the most applicability for comparisons with state monitoring programs. The objective of the surveillance monitoring

component is primarily to enforce U.S. pesticide

residue tolerances established by the U.S. Environmental Protection Agency (EPA). The tolerances are

established by Federal regulation and published in

Code of Federal Regulations 40 Part 180.

Sampling of imported produce is based on the regional import sampling plan, on headquartersdirected assignments, and on special emphasis surveys. In developing regional sampling plans, districts consider the dietary significance and production volume of the commodities, the compliance

history of the country of origin, and pesticides used

at origin identified through use of the Battelle World

Agrochemical Data Bank. Because of resource limitations, USFDA headquarters places some restrictions on the commodities to be sampled based on

the commodities’ significance in dietary intake. For

example, very few samples are taken of spices and

herbs.

Headquarters-directed assignments are aimed at

obtaining residue data for commodities or pesticides that have not sufficiently covered during previous years. Also included in these assignments are

pesticides or commodities that, on a national level,

are of increasing concern or interest. Examples of

headquarters-directed assignments proposed for the

1988 Federal fiscal year include imported fresh cucumbers to be analyzed for organohalogen, organophosphorus, and carbamate residue; and imported

fresh apples to be analyzed for organohalogen, organophosphorus, Ethylene Bisdithiocarbamates

(EBDC), benomyl, thiophanate-methyl, Methyl 2-

benzimidazdecarbamate (MBC) and daminozide

residues,

Special emphasis surveys are based on selected

high volume imports and on commodities treated

with pesticides that are not allowed for food use

in the United States. Each district is required to select and conduct a minimum of two of these surveys with priority given to country/commodity combinations not covered by previous monitoring in the

district (3).

For domestic samples, USFDA districts prepare

annual sampling plans based on local conditions

such as pest problems, amount of production, past

compliance history, or coverage. Headquarters

specifies the minimum number of samples to be

taken by each district and the resources to be expended on pesticide monitoring. In its annual guidance to the districts, the headquarter’s office also

specifics coverage of certain pesticides and commodities for each district. This special-survey element normally focuses on pesticides that are of potential health concern and that require analyses by

single residue analytical methods, or it monitors the

level of specific pesticides of importance to the EPA.

For example, for several years a special survey was

performed of EDB in grains and fruits. EPA needs

information on the extent to which EDB residues

were occurring because of carcinogenic concerns.

Other than specific surveys, districts are given

considerable latitude in developing annual plans for

domestic sampling. Most plans are designed to

cover crops of local dietary importance, pesticides

with high usage within the district, growers or commodities with past compliance problems, and commodity/pesticide combinations in which misuse is

suspected.

Normally, samples are analyzed by one of five

multi-residue techniques that detect from 24 to 123

pesticides. Single residues, or specific analyses, is

performed during special surveys on specific pesticides, to confirm levels detected by multi-residue

techniques, or when misuses of the pesticide is

known or suspected (7).

Florida Department of Agriculture and

Consumer Services (FDACS)

FDACS began monitoring raw agricultural commodities for pesticides residues in 1960. The Bureau of Chemical Residue Laboratory, under the Division of Chemistry, FDACS, is responsible for the

analysis of pesticide residues in food and feed products produced or marketed in Florida. It is also responsible for the enforcement of Federal tolerances

and guidelines adopted by the state. Each year the

Bureau performs more than 10,000 determinations
194

on approximately 4,000 food and feed samples.

Each routine sample is analyzed by the chlorinated

hydrocarbon and organophosphate multi-residue

procedures. Samples of fresh fruits and vegetables

are also analyzed by the carbamate screening procedure. Single residue analyses are performed on

an “as needed” basis. Analyses of the majority of

routine food samples are completed within 48 hours.

Since the late 1970s, the program has been setup

to target the most probable problem areas in order

to direct limited resources. Samples are taken of

commodities throughout the channels of trade, including airports and docks. Samples may be of

Florida-grown or imported produce, depending on

the time of year. Florida’s program is a combined

crop-pesticide index. That is, in selecting samples

for analysis, FDACS considers the propensity of the

commodity to retain significant levels of pesticides,

and the characteristics of the pesticides applied to

the crop. According to W. George Fong, FDACS,

the classification of crop groups from the standpoint

of potential pesticide exposure of consumed plant

parts is based on the book Food and Feed Crops

of the United States, by J.R. Magness, et al. (2). Considerations of the characteristics of the pesticides

applied to the crop include the following: acute oral

toxicity, persistency in the crop, toxic metabolizes

formed, current EPA special review, systemic property of the pesticide, and human dietary exposure

(2).

Massachusetts Department Of Public

Health (MDPH)

MDPH has been performing pesticide residue surveillance for about the past four years. Approximately 500 samples are taken each year that can

be analyzed for about 30 different pesticides. Recently, MDPH has made a rather major change in

its program direction. The department’s program

is tailored to identify and assess specific potential

pesticide-related health risks. Potential risks are

identified either through risk assessment analysis

or through laboratory results. For example, if the

laboratory detects significant levels of a chemical

in food, risks assessments associated with that level

will be initiated; conversely, if risk assessment demonstrates a concern for a particular chemical, the

program will be directed toward analyzing commodities on which that pesticides may be used.

The objective of the program is to identify and

assess the pesticide residues that may pose the greatest risk using information and criteria from the FDA

surveillance index, data on file with the EPA and

with other states (for example, from the FOODCONTAM program, a federally sponsored data-sharing

program that collates pesticide residue analytical

data from the laboratory. Emphasis is placed on the

diets of those subgroups of the population determined to be most at risk from exposure. Samples

are taken at the wholesale and retail level for both

domestic and imported commodities (1).

Montana Department of Food and

Agriculture (MDA)

MDA has been taking pesticide residue samples

for about 13 years. An average of 250 samples are

taken each year as part of agricultural pesticide misuse investigations. An additional 50 samples per

year are specifically collected for residue monitoring (or tolerance enforcement) in food commodities. For misuse investigations, the pesticide analysis is normally limited to the specific suspect

pesticide. For the monitoring program, any or all

of the major pesticide groups are requested, e.g.,

carbamates, organophosphates, etc.

The majority of samples consist of agricultural

commodities produced and marketed in the state

that are known to have been treated with a specific

pesticide. This normally occurs after a pest outbreak

that has required extensive applications of the target pesticide. Samples are taken at the farmgate or

retail level. The analytical laboratory is capable of

analyzing for 70 to 100 different pesticides both

through multiresidue and specific analyses. Analyses requested are dependent on the situation triggering the sampling.

California Department of Food and

Agriculturo (CDFA)

CDFA has had a pesticide residue program for

more than 60 years. CDFA’s pesticide residue monitoring program is organized into four major components: state routine, preharvest monitoring, focused

monitoring, and processing foods monitoring. Altogether, the California program results in more

than 43,000 determinations on approximately 13,000

samples each year. These samples are in addition

to samples analyzed during misuse investigations,

which account for an additional 4,000 samples per

year. The state routine component is a commoditybased, tolerance-enforcement function consisting

of approximately 6,500 samples of fresh fruits, nuts,

and vegetables taken from throughout the channels

of trade. Both domestic and imported commodities

are included. Analysis for the majority of these samples is by multiresidue screens, capable of detecting approximately 100 pesticides, Analyses per-
195

formed through multiresidue screens are normally

completed within 4 to 6 hours from the time the

sample is submitted to the laboratory. For this component, single method analyses are made on an “as

needed” basis, with a turnabout time of generally

less than 24 hours. The selection of 75 percent of

the commodities sampled in this component is

based on a statistical formula that takes into account

the amount of consumption and historical residue

data. Specialists are allowed to use their discretion

in selecting the remaining 25 percent. Factors influencing discretionary sampling include knowledge of pest problems and pesticide usage within

the production areas, data from the USFDA program, etc.

The preharvest monitoring component consists

of approximately 2,500 samples taken from fields,

prior to harvest. These samples are normally analyzed by the multiresidue screens. Specific analyses are requested on an “as needed” basis. Commodities to be sampled are determined by the

amount of production in the county of origin, pest

problems, pesticide usage within the production

area, and by compliance history of the grower. Early

detection and deterrence of pesticide misuse is one

of the major goals of this program.

The focused monitoring component is a pesticidebased, rather than commodity-based, program.

Each year, CDFA medical toxicologists identify pesticides of priority health concern. Commodities

known to have been treated with those pesticides

are sampled and analyzed for the specific pesticide.

As with the Massachusetts program, emphasis is

placed on the diet of those subgroups of the population determined to be most at risk.

The processing foods monitoring component consists of approximately 1,500 samples of raw commodities destined for processing. Samples are taken

in the field, shortly before harvest or after harvest;

at grading stations; and at processing plants prior

to processing. These samples are analyzed by multiresidue screens. An important goal of this component is to provide information to the California Department of Health Services (CDHS) to assist them

in designing their processed-foods-products pesticide monitoring program. The number of samples

to be taken of each commodity is based on California production figures.

As can be determined from the previous discussion, there is quite a variety in the types of samples, types of pesticide analyses performed, and

sizes of state programs. The objectives of state programs also vary dependent on resources and public concerns. However, similarities also occur. For

example, the Massachusetts program, which is

limited to 500 samples per year, has chosen to focus its sampling on specific pesticides as they relate to dietary risk. Though similar in size, this program is similar to California’s focused monitoring

program. The same theme can be seen in USFDA’s

headquarters-directed and specific emphasis assignments.

In Montana program, resource limitations have

caused this state to restrict its monitoring solely to

those situations in which the possibility of overtolerances is the highest. The Florida program,

though larger, has also directed its program in this

way.

Most of the programs are, at least partially, developed to act as a deterrent to pesticide misuse.

The California program, however, is the only one

that routinely takes samples of commodities in the

field prior to harvest, as well as in the channels of

trade. The Massachusetts program appears to be

based more on public health concerns than on deterrence.

All program have multiresidue screening capabilities. There is variation, though, in the number

of pesticides that can be analyzed in this manner.

Further discussion on analytical capabilities will

follow.

All states contacted have the authority to adopt

their own residue tolerance levels; however, all of

them currently use those set by EPA. USFDA and

some states also use “action levels” and “regulatory analytical limits” in determining whether or

not to take enforcement action. The use of action

levels and regulatory analytical limits is not uniform.

A recent decision by the U.S. Court of Appeals

for the District of Columbia stated, in essence, that

action levels set by USFDA were legislative rules

rather than general statements of policy and, therefore, must be adopted according to the Administrative Procedures Act. The court found the Federal action levels to be invalid because they were

not adopted according to this procedure. At best,

action levels are useful as a guide and do not require or prevent USFDA from taking action (8).

The results of this 1987 decision have yet to be

fully addressed. In Florida, where no tolerances or

action levels exist for a pesticide in a particular commodity, a regulatory analytical limit is applied (9),

Action levels are treated the same as tolerances. In

cases in which no tolerance or action levels exists,

Florida set its own regulatory analytical limited

based on the lowest residue level the laboratory is

able to reasonably detect, measure, and confirm

with existing analytical methods (2). Historically,
California has acknowledged those Federal action

levels as published in 40 CFR 180, but it has not

acknowledged regulatory analytical limits set by

Federal policy. California is currently re-examining

whether or not action levels can continue to be used

because of the Court of Appeals decision.

Analytical Methods Needed

In the past ten years, the need for and ability of

pesticide residue laboratories to identify, quantitate,

and confirm the presence of trace levels of pesticides in or on food crops has increased dramatically. California regulations require that the

pesticide-residue analytical method submitted in

support of a California registration for food-use pesticides not exceed 24 hours. The Florida enforcement program’s mandate allows for food samples

to be completed within 48 hours (2). The EPA currently has a guideline for analytical methods that

emphasizes the desirability of a 24-hour method,

but it is not mandatory.

Multiresidue screens currently being used by

states are useful; however, they are not inclusive.

Some pesticides do not lend themselves to a screening procedure because of their chemical constituents. Others, though they can be detected in water

samples, require extensive preparation time for detection in the various crop matrices. When there

is need for data on a non-screenable chemical, the

slower single residue analytical method(s) submitted

by the registrant or a PAM method must be employed.

There is a need for more multiresidue procedures

that detect metabolizes as well as the parent compound. For tolerances enforcement programs, time

is of the essence, and analysis should be completed

within a normal working day, making multiresidue

screens ideally suited for this type of work. Many

single residue methods also meet this criteria, although in some cases laboratories must modify submitted methods to achieve this time frame. Built-in

quality-assurance features are needed, and methods should not require specific instrumentation that

only a few state laboratories have or can afford (2).

Performance characteristics of the ideal analytical methods for pesticide residue in food crops

would have the following minimum characteristics:

1. Methods would be validated on every crop type

for which the pesticides is registered. As new

registered food crop uses are approved, the analytical methods would be updated to reflect the

new crop matrices. For example, an analytical

method may be acceptable in selectivity and

sensitivity for head lettuce, but when the same

analysis is performed on green onions or parsley, the crop matrix interferences may reduce

the analytical sensitivity to an unacceptable

level.

2. All new analytical methods would be validated

in a series of independent laboratories. This

procedure would test the method to evaluate

its reliability and reproducibility under various

operating and management systems.

3. New methods or analytical regimes would have

to include the ability to detect, identify, confirm, and quantify and and all metabolizes included in the 40 CFR 180 tolerances. Ideally,

this process should not exceed seven hours

from the time the sample is received in the laboratory.

In addition to developing methods for new chemicals, review should be completed on the older

chemicals, especially those with potential dietary

impact. For example, the current approved methods for EDBC’s are not product specific, and there

are no known confirmational techniques. The only

approved method is wet chemistry and involves CS2

evolution and calorimetric quantification. Besides

the obvious shortcomings of these types of methods, different tolerances exist for the various members of this family of chemicals on the same crop.

There is no way, short of field investigation, to determine which tolerance applies and if an overtolerance has occurred.

The needs of the pesticide regulatory programs

for accurate data demand that the laboratories monitor their ability to provide accurate, timely, and reproducible analytical results. In order to assure

these results, use of a well-managed quality-control

or quality-assurance program is needed. In most

states, such a program has been initiated. However,

there is a need for development of new analytical

quality-control methods with internal provisions,

These internal checks could alert the analyst to developing problems and the need to effect timely corrective action. Such a system could greatly reduce

the time currently being spent to investigate the

causes of inaccurate analytical results, thereby reducing the analytical cost per sample.

Many of the newer pesticides being used on food

crops are thermally liable and not easily analyzed

by the high temperature GC systems. The other major analytical tool widely available for use is HPLC.

The HPLC, however, lacks easy or reliable analytical confirmation. New methods that will provide

quick, reliable, and cost-effective confirmation that

will also be legally defensible are needed.
197

For pesticide residue enforcement, analytical

methods that are specific for the parent chemical

are needed. Currently, EPA does not require specific analytical methods for the parent compound.

Metabolizes included in the tolerances listed in 40

CFR 180 need to be identified. Currently, there are

tolerances that state “and cholinesterase-inhibiting

metabolizes” or “and its metabolizes” (40 CFR ed:

1980). Confusion exists regarding what parts of a

commodity must be included in the analytical procedure. It is imperative that EPA of FDA take action to relieve this confusion. Suggestions would

include publishing a single-source document such

as that included in CODEX that provides this information or establishing a toll-free telephone number to an information officer to answer questions.

This information center should be staffed around

the clock to be of service to all states.

To facilitate detection of pesticide misuse, there

is a need for development of residue analytical

methods for various agricultural and environmental

media, and for crops for which the pesticides in

question are not necessarily registered. According

to Laszlo Torma, Montana Department of Food and

Agriculture, the methods in the PAM II are inadequate because they are not collaborated, and they

are designed only for those commodities for which

the chemical is registered. Companies and Federal

laboratories with the assistance of state laboratories

could set up and collaborate multiresidue methods

for these compounds, and special consideration of

a region could be acknowledged to meet these methods and regulations. For example, in Montana a

relatively large number of the population consumes

meat from wildlife on a regular basis; however,

there is no official collaborative analytical method

or established tolerance available for these foods.

Another area that could be addressed is the pesticides that are not registered in the United States,

but are registered in Canada. Frequently, these

products enter Montana and other bordering states

but when residue analyses are required, there is no

method available (6).

Emerging techniques such as immunoassay and

biosensors have potential for pesticide residue analysis. The initial impact of these new techniques is

expected to be in the area of rapid screening of produce samples for a wide range of specific pesticides.

Under this approach, the confirmation of the screening results would be via traditional GC, GC/MS,

LC/MS, or other appropriate separation and confirmation systems. As the new techniques are

proven to be accurate, dependable, and have internal quality-assurance checks, the classical confirmational steps could be reduced.

The major advantage of these new technologies

would be their improved sensitivity and selectivity.

CDFA is currently evaluating three ELISA techniques for use in the pesticide residue program.

Two of the ELISAs are for the triazine class of compounds and one is for paraquat. The paraquat

ELISA is of interest because it is potentially superior

in sensitivity, selectivity, and reproducibility to the

existing battery of available calorimetric methods.

Evaluation at CDFA indicates that the new technologies are rapid, reproducible, and inexpensive

to use. These factors open the possibility of regulatory programs being able to perform more analyses per sample and to run more samples for selected

pesticides. This would enhance the regulatory data

base and provide statistically valid residue trends

and dietary loads.

These new technologies appear to be “user

friendly”, and the amount of time and money to

train staff to utilize these systems appears to be minimal. These procedures are “turn-key”, and any laboratory could improve its capability without a massive infusion of funds.

At this time, however, most of these methods are

qualitative, or at the most semiquantitative procedures. They do not promise, however, for being

used as a preliminary screen (2). Research and field

testing should be given high priority to make these

tools available within the next few years.

Analytical Methods Developed as

State Level

Analytical methods development at the state level

varies with the objectives of the various state programs. Most state programs are primarily focused

on enforcing Federal tolerances. To be effective, this

type of enforcement requires rapid turnaround

time. This often necessitates modifying existing analytical methods or developing a new analytical

method, For example, CDFA laboratories have

adapted a more rapid GC method for EBDC’s in

place of the Federal wet-chemistry method to be

compatible with states’ regulatory needs.

States that actively investigate pesticide misuse

and pesticide illness incidents often must modify

residue methods to meet their needs. For example,

CDFA has modified analytical methods that were

developed for food crop analyses to be applicable

for different analytical uses, such as farmworker

exposure monitoring or environmental drift and

contamination of non-target areas.

Florida’s laboratory has developed an HPLC-UV

screening procedure for several families of herbi-
198

cides, for example, triazines, uracils, phenylureas,

etc., in water samples. When the sample preparation technique is worked out, these procedures can

be used for vegetable and fruit samples as well (2).

In Massachusetts, EPA’s decision not to ban the

use of daminozide caused concern at the state level,

and Massachusetts decided to take independent action. In order to perform the analytical testing of

raw and processed apple products necessary to

complete their risk assessment, this state’s laboratory developed analytical methods to improve the

sensitivity levels (l).

CDFA’s resources for analytical methods development are devoted to modifying existing methods.

Currently, the State of California may have the

largest state-funded pesticide analysis program

among states, CDFA’s Chemistry Laboratory Services branch has a methods-development group

staffed with one principal agricultural chemist

(Ph.D.), two agricultural chemists III, which is the

highest technical pay-grade in the state’s system,

and one experienced technical assistant. As part of

the methods-development group, an in-house

quality-control and quality-assurance program has

recently been initiated and maintained by an agricultural chemist III and a technical assistant.

CDFA is involved in the evaluation of new analytical technologies such as the applications of super critical fluid chromatography in pesticide residue chemistry, ELISA, and tandem mass spectrometry

through the methods-development group. Due to the

geographical location and the past close working

relationship with the University of California at

Davis (UCD), CDFA is exploring the possibility of

a state-funded collaborative effort for analytical

methods-development research with UCD. This effort could include the following: (1) improvement,

modification, and unique application of conventional analytical methods, for example, GLC, LC,

and wet methods; (2) nonconventional analytical

methods development, for example, ELISA, alternative detection of pesticides, novel separation science; (3) confirmation of analytical results through

shared advanced instrumentation facilities, for example, HR MS, MS MS, Foumer Transfer Infrared

Spectrometer (FTIR), and Nuclear Magnetic Spectrometer (NMR); and (4) training of appropriate personnel and technology transfer.

In the area of instrumentation review, California

is currently looking for a better and more reliable

confirmational technique for GC and HPLC systems. Currently, California is evaluating a

GC/MS/LC for both GC and HPLC work. A GC Mass

Selective Detector (MSD) will be purchased for

evaluation along with further work with photodiode array detectors and supporting work stations.

This type of work is very expensive for a state to

fund and it is, therefore, limited in scope. The work

conducted in California’s laboratories is focused on

addressing California’s needs and may, therefore,

not be of any utility to other regulatory or commercial pesticide residue laboratories.

In Florida, methods-development work has traditionally emphasized modification of existing techniques. To augment existing methods, this state is

now extensively using Solid Phase Extraction (SPE).

According to W. George Fong of FDACS, “SPE technique for sample preparation requires less sample

and solvents and can be completed in much less

time. It reduces the health hazards in the laboratory and generates less solvent waste. SPE also provides limited specificity” (2). FDACS has developed

SPE techniques for carbamate analysis and adapted

the techniques for most HPLC analyses. Some

limited preliminary studies are also being done of

SPE for gas-liquid chromatography. There are two

chemist positions devoted to methods development

and quality-assurance work in this state.

In Montana, methods-development is limited to

determining the accuracy of a published method

or adapting a published method for a given commodity to another commodity. Local needs further

limit methods development primarily to areas of

new herbicides (glyphosate, sulfonylurea herbicides, triazin, substituted ureas, etc.). This state has

three chemists who expend approximately 70 percent of their times on residues analyses.

In Massachusetts, methods-development work

has been on a case-by-case basis. This has involved

pesticide-specific surveillance and compliance testing for chemicals that have been designated as public health priorities and have required state-level regulatory action. During 1987, activities include risk

assessment and policy development for alachlor and

2,4-D, methods development and compliance testing for daminozide in apple products, and screening for heavy metals and organochlorine pesticides

in bottled drinking water.

There is undoubtedly some duplication of analytical methods-development efforts by states and

Federal government agencies when the objectives

of the programs are similar, for example, monitoring for tolerances enforcement. In the past, little

or no information was exchanged between state laboratories and Federal agencies regarding research

or methods-development work being conducted.

Currently, CDFA and USFDA Region IX are developing a memorandum of understanding (MOU) that
199

will include a residue analytical section. Methodsdevelopment and quality-assurance procedures are

being considered to be included in this MOU.

Impact of Local Regulatory

Initiatives

State laws and regulations can place special analytical requirements on pesticide laboratories. Several examples from California illustrate this point.

Historically, California law has provided that CDFA

may seize a lot of produce if it is suspected of carrying excess pesticide residue. By statute, the lot

may only be held for 24 hours unless laboratory

analysis confirms the existence of the illegal residue. This has caused CDFA to modify or replace

methods that take longer than 24 hours to complete,

Proposition 65, passed by the California voters in

1986, provides that no person shall expose any individual to a chemical known to be a carcinogen

or reproductive toxin without giving prior warning. While all of the ramifications of this law are

yet to be understood, it is conceivable that the pesticide regulatory laboratories will have to modify

or replace existing residue methodology to shorten

turnaround time or to lower limits of detection for

pesticides that are oncogenic or have adverse reproductive effects.

Recently, commercial laboratories in California

have begun to conduct pesticide residue testing on

produce for grocery stores. A bill has been introduced in the state legislature that would require

such laboratories to be accredited by the state and

to participate in a state-sanctioned quality-assurance

program. Accreditation programs may place additional responsibilities on state laboratories to provide oversight, oversee and qualify control of uniform analytical methods.

Neither Florida nor Montana reported having legislation similar to Proposition 65, though Florida

does have a regulation that suspends the use of aldicarb in an area where it has been found in well

water in excess of 10 parts per billion. Massachusetts, with program emphasis on pesticides with potential chronic risks, may well have occasion to employ modified residue methods.

Role of States in Analytical

Methods Development

Pesticides analytical methods-development at

state laboratories has a different focus than that of

academia, private industry, and Federal government agencies, Rather than the development of

basic new analytical methods, state laboratories emphasize methods-application and subsequent

methods-modification. This difference in emphasis has arisen from differences in the overall objectives of the various laboratories. Traditionally,

academia has contributed in the aspects of basic,

novel analytical methods-development, while industries have emphasized analytical methods for applications of a particular chemical. In general, state

laboratories’ needs for pesticide analytical methods

are to monitor, regulate, and enforce the uses of pesticides within a given state, in accordance with

federally-established standards, However, there are

still differences in analytical needs between Federal and state laboratories.

The historical and current role of California in

analytical methods research and development has

largely been limited to modification of existing

methods to meet our criteria of performance acceptance. There have been instances when new methods have been developed for residue analysis because existing approved methods were not specific,

rapid, or sensitive enough to meet regulatory needs.

For example, in 1985, contamination problems resulting from the misues of aldicarb on watermelons

resulted in a recall of all California watermelons.

In order to allow continued sales, California established a certification program that included sampling and analysis of melons, from all fields prior

to shipment, for aldicarb and aldicarb sulfoxide. The

original method was judged too time-consuming,

as each of the four CDFA district laboratories was

attempting to “clear” 20 fields per day, and each

field required a minimum of five composite samples. With the single goal of certification in mind,

an HPLC method was developed to provide the accuracy speed, and precision required (5).

In addition to ongoing methods modification,

CDFA’s methods-development group is addressing

the use of new instrumentation technology in various residue applications, The pesticide registration

laboratory works with pesticide registrants in order to resolve operational problems with their analytical methods. This activity is restricted to

methods-modification, not conducting research,

which is the responsibility of the pesticide registrant. Work conducted in CDFA’s laboratories focuses on addressing California’s needs and may,

therefore, not be of any utility to other regulatory

or commercial pesticide residue laboratories.

In the area of methods-development, Federal

agencies should seek states’ input to determine what

the state’s analytical needs are. Collaboration is nec-
200

essary between state, Federal, and private laboratories. Additionally, states with smaller programs

would benefit from a more vigorous training program provided by Federal agencies (6).

Massachusetts sees a need for FDA training programs for pesticide analytical laboratories. Training is necessary for comparability and accuracy of

data, including analytical support and guidelines

for data interpretation for a variety of analytical procedures and instrumentation. Also necessary, from

this State’s viewpoint, is the establishment of minimum criteria by which a laboratory would be considered a certified FDA laboratory. Such a program

would include quality-control and quality-assurance,

possibly including specified recovery rates and detection limits,

In order to provide uniform regulatory analytical results and assure consumer protection, consideration should be given to an EPA/FDA/NBS/statemanaged laboratory accreditation program for all

pesticide residue regulatory laboratories. As part

of this program, EPA/FDA should conduct quarterly

regional meetings with the state laboratories and

a national meeting for all state chemistry managers.

These meetings at the local and national level would

provide state input into the national programs.

Many new pesticides are on the horizon which

will require very different types of analytical methodology than are currently utilized in state programs. What role will states play in developing/modifying methods to analyze these pesticides? Will

state laboratories be able to maintain the efficacy

of their programs through modification of existing

techniques? State programs have no role in developing analytical methods of support the registration of a pesticide. The responsibility to provide an

acceptable analytical method at the time of product registration is clearly that of the registrant,

whether that method is a modification of existing

techniques or development of a completely new

type of methodology, The role of states will continue to be working on published methods to improve their sensitivity, expanding the types of sample matrices, and optimizing registrants’ methods

for use in the state’s analytical system.

The role of states in developing/testing new methods such as immunoassay, automation, and screening will be different depending on the size of the

state’s programs and the available funding. CDFA

has defined its role in the development and testing

of newer methods to include the identification of

analytical needs, both short-term and long-range;

and contractual support for development, or cooperation in product evaluation, such as with private

immunoassay product suppliers. The use of automation and the development of expanded or new

multiresidue pesticides screens are part of an ongoing process in California’s program. CDFA is actively engaged in reviewing its analytical procedures for incorporation into an automated system,

and expects to test a robotic system within the next

18 months. The expansion of current multiresidue

pesticide screens and the development of new

screens are priorities for CDFA’s method development group.

The role of state pesticide residue monitoring is

to supplement the broad Federal program, while

focusing activities on crops produced within state

boundaries. Cooperation is essential to minimize

duplication of analytical methods-development. The

Federal agencies responsible for food safety must

provide the leadership in any cooperative effort.

This leadership role must be open to address the

real needs of the states and be sensitive to local conditions. The cooperative effort should include the

development of a national set of methods-development goals that, in turn, could be monitored to ensure efficient use of resources. The technology sharing would reduce duplicative work and encourage

state involvement in problem resolution,

Each state may have different analytical requirements and resources; however, there is common

ground. On role that states could play would be in

an advisory capacity to the Federal government.

The establishment of a methods research and development advisory committee to the appropriate

Federal agency should be encouraged. Such a committee would include representatives from the

state’s chemistry laboratory programs, along with

representatives of consumers, production agriculture, academic institutions, pesticide registrants,

and Federal programs. This advisory group could

be composed of two subgroups: one to deal with

policy issues, which would include the chemistry

program administrators; and one to deal with scientific issues, which would include the principal

chemists. This advisory group could be mandated

to advise the Federal government on current problem areas, results of on-going state-sponsored

projects, and recommend areas of research.

1.

References

Chung, C., Weiss, L., Nassif, J., Ridley, N., Timperi, R., “Planning a State Program for the Surveillance of Pesticide Residues in Food, ” Presentation at the 1987 American Public Health

Association Meeting, October 1987,
201

2. Fong, W. George, personal communication, 1988.

3. Food and Drug Administration, Compliance Program Guidance Manual, Pesticides and Industrial

Chemicals in Imported Foods, (FY 88), 1988.

4. Gingery, G., personal communication, 1988.

5. Ting, K. C., Kho, P. K., “High Performance Liquid Chromatographic Methods for Determination

of Aldicarb Sulfoxide in Watermelons, ” Bulletin

of Environmental Contamination and Toxicology

37: 192-198; 1986.

6. Torma, L., personal communication, 1988.

7. United States General Accounting Office, Pesticides, Need to Enhance FDA Ability to Protect

the Public from Illegal Residues, October 1986.

8. Wessel, John R., personal communication to R.V.

Peterson, 1988.
202

Pesticides AnaIytical Methods Development in the

Private Sector

Lyle D. Johnson, Analytical Bio-Chemistry Laboratories, Inc., Columbia, MO

and

John E. Cowell, Monsanto Agricultural Company,

Life Sciences Research Center, Chesterfield, MO

Contents

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . i

Overview of the Private Sector . . . . . . . . . . . . . . . .

Pesticide Residue Analysis . . . . . . . . . . . . . . . . . . . .

Emerging Technologies . . . . . . . . . . . . . . . . . . . . . . .

Multiresidue Technologies . . . . . . . . . . . . . . . . . . . .

Federal Interaction. . . . . . . . . . . . . . . . . . . . . . . . . . .

Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~

Environmental Sciences Department,

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As a result of questions sparked by the ability to

measure chemicals at increasingly minute levels,

there has been an increased interest in the development of analytical methods for the detection of

pesticide residues in foodstuffs. Among those in the

private sector, most laboratories involved in pesticide method development have typically been university, industry, and contract laboratories. Food

producers, food processors, and distributors also

have an interest in analytical methods.

This discussion will focus on the objectives, the

driving forces behind development, and the ramifications of these analytical methods. In addition,

assessment of existing and emerging technologies

will be performed from a private-sector viewpoint.

Viewed constraints and opportunities will beaddressed together with possible approaches to enhancing multiresidue method development, This

multiresidue screening approach is necessary from

an expedient and cost-effective perspective.

Pesticides have evolved over the decades from

persistent, long-term control, broad-spectrum efficacious chemicals toward short-term control, biodegradable chemicals used with integrated pest

management practices. The resulting agencies have

required the manufacturers to do extensive screening for toxicological and ecological concerns in the

development of any new pesticide, Beyond requirements, each segment of the agricultural industry,

whether it be grower, food producer, distributor,

manufacturer of agricultural chemical, or regulating agency, has increased interest in the issue of

pesticide residues in food.

A tremendous amount of expertise for analytical

methods development exists in the private sector.

The value of this actual experience in developing

methods for the analysis of pesticide residues is

often underestimated. The goal of this residue methodology development effort, whether the laboratory

is a university, pesticide industry, food producer,

food processor, or consulting contractor, is basically

the same: to answer the question of how much residual pesticide is contained in the matrix of interest, The incentives and extent of participation of

the different types of laboratories vary.

Overview of the Private Sector

The university laboratory may perform method

development for the sake of knowledge and achievement, while the food producers and food processors are only concerned that the screening methods used, assure their products contain less

pesticide residues than the corresponding tolerance

(maximum allowable) levels, These tolerances,

which are granted under the Federal Food, Drug

and Cosmetic (FDC) Act, are established from su-
203

pervised field trials at locations representative of

each of the major crop-growing areas. The residue

field trials are conducted by the pesticide registrant

under the most extreme conditions of proposed use,

such as the maximum application rate, the maximum number of applications, and the shortest interval from application to harvest. This measurement process ensures that the tolerance levels,

established under the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and regulated

by the Environmental Protection Agency (EPA), are

not exceeded. The Food and Drug Administration

(FDA) conducts a program of monitoring for pesticide residues primarily in raw, unprocessed food

moving in the commercial channels of trade. Thus,

the obvious interest on the part of the food producers is to ensure compliance.

Section 409 of the FDC Act, adopted in 1958,

established the procedure for tolerances for processed foods and animal feed when pesticide residues on the raw agricultural commodity (RAC) concentrate in a processed fraction of the RAC. For

instance, when raisins are processed from grapes,

if a concentration of a residue occurs, then a foodadditive tolerance is required for the pesticide in

raisins. Conceivably, this concentration could make

an undetectable residue in the RAC, detectable in

the processed food. Some pesticides of toxicological concern concentrated in processed food would

trigger the Delaney Clause. Food processors perform analyses to assure that their processed products contain undetectable residue levels or levels

less than these food-additive tolerances.

Several contracting laboratories were surveyed

(1, 2,3, 4,5) to determine their involvement in pesticide analyses and the level of methods development. Contract laboratories obtain analytical procedures from their clients, peer-review organizations,

literature references, or when not available, develop

a procedure from innovative research. In the cases

of FIFRA registration projects, analytical methods

are generally provided by the registrant. At best,

research will be limited to adaptation of a method

for additional sample matrices. Those laboratories

concerned with minor-use pesticide registration,

such as the regional IR-4 laboratories, respond in

much the same way as a contracting laboratory. A

method is provided by the company sponsor and

used to acquire registration data (6). These IR-4 laboratories are usually affiliated with universities.

These university laboratories have the analytical

residue and method development experience and

perform very cost-effective residue analyses. Other

university laboratories have not shown a consistent interest in residue analysis except as an application for specific analytical techniques.

The contracting laboratory strives for a competitive edge by analytical method development for a

purely monetary interest, while the industrial pesticide laboratory has the weight of economic and

social responsibility to comply with the regulatory

requirements for EPA registration. In addition to

the development of residue field-trial data for estimation of the tolerance level, the pesticide registrant conducts reproduction and long-term animal

feeding studies, using various species of test animals to establish the safety of the tolerance level.

These toxicity studies determine the No Observable Effect Level (NOEL), the level at which the pesticide has no harmful effect on the most sensitive

test animal. This NOEL is divided by a safety factor of up to 100 or more to set the Acceptable Daily

Intake (ADI). The ADI represents the amount of pesticide residue that can be ingested by an average

person every day for a lifetime without ill effect,

Thus, the ADI usually is less than the sum of the

normalized tolerances of the pesticide residue levels

for all registered uses on crops.

The first step in the process of developing the crop

residue and the environmental fate data base for

a pesticide is the development of the analytical

method. Subdivision O Residue Chemistry Guidelines (7), developed by the EPA, state that the pesticide registrants need to develop methods for residue analyses that serve two functions: 1) they must

provide the residue data upon which judgments are

made as to the identity and magnitude of residues

from the proposed use, and 2) they must provide

a means for enforcement of the tolerance. Sometimes, these two functions are best served by development of two separate methods. The initial role

of developing analytical methodology justifiably belongs to the pesticide manufacturer. In addition to

the production of the parent active ingredient of

the pesticide, the manufacturer has had to synthesize the degradants or metabolizes for identification

purposes as well as for reference standards for the

residue method development. The manufacturer is

in the best position to develop data on the product

chemistry, physical properties, and means of analysis of the pesticide.

As is inferred from the EPA guidance document,

there are two driving forces in the development of

analytical methodology. One is to develop an analytical method to provide data to quantitate the magnitude of residues from the proposed use to establish the residue tolerance, Toward this goal, there

is a certain amount of pressure on the industrial
204

chemist to develop methodology as quickly as possible, given that it takes approximately 5 to 7 years

to perform the necessary toxicological, environmental fate, metabolism, and residue studies to fulfill regulatory requirements to ensure registration

of a product to allow it to be sold. Thus, any time

saved in this process or timeline can result in market entry advantage and greater profitability. During this rapid development, the optimal method

speed and universality is not always addressed. The

developing chemist is concerned mainly with quantitation of all required substances, with method sensitivity in that the method must be capable of detecting very low levels (i.e., an acceptable low level

in food and feed matrices would be 10 to 50 ppb)

and with method selectivity in that there are no interferences that would result in false-positive detections with use of the method. Thus, to achieve

this high degree of sensitivity and selectivity in the

most rapid fashion, the developer is compelled to

use the most powerful state-of-the-art analytical

techniques and instrumentation available. Additionally, the relatively large number of analyses needed

to support a registration submission further serves

as validation of this analytical methodology.

From a contract laboratory standpoint, the pressure to stay on schedule analytically with the various ecological, environmental fate, and residue

chemistry studies is overwhelming. The registrant

can be a very demanding sponsor as a result of the

timeliness desired or imposed by the EPA. Methods are sent to laboratories for validation in both

tested and untested matrices. Quite often, these

methods have not undergone ruggedness testing to

identify the critical steps of the procedure. Ruggedness testing through collaborative interlaboratory

study determines the reliability of each step of the

method by performance by several different

analysts. Most laboratories have experienced, to the

detriment of the analytical method, undesirable

levels of method variability with different lots of

reagents, absorbents, and column materials.

The other driving force is to develop a method

that can be used to enforce the established tolerances. This methodology is usually different from

the previous method because it has to be as simple

as possible to minimize the cost of monitoring for

pesticide residues. The EPA required enforcement

method is expected to be rapid (less than 24 hours

to completion), sufficiently sensitive in relation to

the tolerance, interference-free, free of blanks or internal standards, and unencumbered by exotic

equipment or reagents. The use of multi-detection

methodology is extremely desirable. However, the

method must measure the “total toxic residue”, as

determined in the metabolism studies [171-4(a), Nature of the Residue]. This total toxic residue includes

the parent molecule and all metabolizes of toxicological concern. Since most metabolizes are not

tested for toxicity, this means all metabolizes isolated in sufficient quantities to be identified. This

requirement greatly increases the level of difficulty

in the development of multiresidue methodology

and will be explained later in this discussion.

Pesticide Residue Analysis

Pesticide residue analyses can be classified into

three groups for the purpose of examining applicability to multiresidue analysis:

1. compounds that do not degrade or metabolize

quickly,

2. compounds that do degrade or metabolize

quickly, and

3. compounds that degrade or metabolize at a rate

that falls between these two groups; they are

degraded to only a couple of additional compounds.

Compounds that are not degraded or metabolized

either rapidly or significantly in the various environmental compartments (i.e., air, soil, water,

plants, or animals) offer the best opportunity for

multiresidue analysis. Only the parent molecule has

to be isolated from the matrix for quantitation. Four

general multiresidue methods for pesticide residues

have been published by the Association of Official

Analytical Chemists (AOAC) (8). These methods

analyze for organochlorine, organophosphorus, fumigant, and carbamate pesticides. Examples of organochlorine pesticides that are addressed by the

multiresidue method are as follows: dieldrin, heptachlor, DDT, lindane, methoxychlor, perthane, aldrin, endrin, and mirex. Organophosphorus pesticides that are addressed include the following:

diazinon, ethion, malathion, methyl parathion,

parathion, and fenchlorphos. Several fumigants are

addressed: trichloroethylene, ethylene dibromide,

chloroform, and carbon tetrachloride. And carbamate pesticides that are addressed are as follows:

carbanolate, carbaryl, carbofuran, and propoxur.

Most of these compounds are very stable, quite persistent, and tend to bioconcentrate in biological media or exhibit cholinesterase inhibition. Unfortunately, these descriptors coincide with what many

feel to be environmentally obnoxious properties.

From an environmental standpoint, one would prefer a pesticide that would degrade or metabolize

quickly to naturally occurring compounds.
205

Compounds that are extensively degraded or rapidly metabolized in the various compartments (i.e.,

no parent molecule remaining) offer the least opportunity for multiresidue analysis. For these pesticides, the most prevalent method-development approach is to convert the multiple degradates or

metabolizes to a common chemophore for quantitation. An example of this is substituted anilinebased products such as diuron, neburon, and linuron, in which analysis is achieved by hydrolyzing

metabolizes to the common 3,4 dichloroaniline moiety. Thus, a multiresidue method using this approach could not address these three pesticides,

since this procedure could not distinguish which

one of the three produced the residue. Additionally, these types of conversion methods need specific optimization of each reaction (i.e., acid, base,

or enzyme hydrolysis, oxidation, reduction, etc.).

For instance, in the previous hydrolysis example,

reaction conditions must be developed to maximize

the yield of 3,4 dichloroaniline from diuron residues

in a crop matrix to achieve the EPA minimum

acceptable recovery of 70 percent. These conditions

may be different for the reactions needed to obtain

an acceptable recovery of 3,4 dichloroaniline from

linuron residues in the same crop matrix not to mention in different crop matrices. Thus, pesticides that

are extensively degraded or metabolized would

probably not be suitable for multiresidue methods.

As a general rule, the larger the number of metabolites, the more difficult the residue method development and the less likely the method would be able

to measure many different pesticides.

Some compounds fall in between the two previously mentioned categories; they are somewhat

degraded or metabolized to only a couple of additional compounds. These pesticides offer some hope

for multiresidue analysis provided, that they are

similar enough to other pesticides and they do not

have common degradates or metabolizes. For future

convenience, metabolizes will be referred to as

degradates. These pesticides and degradates may

not be amenable to direct detection because the

degradates usually contain more polar functional

groups, which require a modified analytical approach, than those used with the parent molecule.

In these cases, the chemist uses chemical derivatization of the degradate(s) to convert them to a more

measurable moiety. Derivatization reactions such

as esterification, acetylation, acylation, silylation,

and many others are used to improve the sensitivity, selectivity, or chromatographic behavior of the

compound. Part of the difficulty in development of

this type of method is in the isolation of the components from as much of the matrix as possible. This

goal is important in order to have the derivatization reaction more closely approach the optimized

“neat reaction” with standard materials. The remaining matrix components could be considered

to quench or in some cases compete with the derivatization reaction and thus lower recovery (yield).

Thus, pesticides that require derivatization don’t

seem to fit as nicely with the concept of multiresidue

methods due to the potential presence of competing reactants both from the matrix and other pesticide residues. This is not to say that with considerable developmental effort a multiresidue method

could not be developed, but many parameters would

have to be explored in such an endeavor.

Existing methods for detecting pesticide residues

in foodstuffs can perhaps best be explained by

breaking the method into two parts: 1) isolation from

the food or crop matrix, and 2) the detection of the

pesticide residue. Isolation of the residue is begun

by solvent extraction of a solid food or by liquid

partition extraction of a liquid food with a solvent

for which the pesticide residue has a greater affinity,

Thus, the residue is removed from the majority of

the matrix components. However, numerous chemical compounds that are components of the matrix

itself are co-extracted, and this is usually the most

difficult part of the analytical method commonly

known as the cleanup. These co-extracted compounds in fact possess properties similar to those

of the pesticide residue and thus are more difficult

to remove. Some of the usual cleanup techniques

employed in analytical methods are the following:

filtration, solvent-partitioning, absorption chromatography, ion exchange chromatography, solidphase extraction, gel permeation, dialysis, and distillation. These techniques are all aimed at the

removal of coextracted matrix materials from the

sample extract. After the cleanup in many cases,

chemical reactions have been used to convert residue components to a chemophore for enhancement

of detectability, specificity, or improved separation

from remaining components.

Two of the most common analytical instruments

for the detection and quantitation of pesticide

residues are the gas chromatography (GC) and the

high-pressure liquid chromatography (HPLC). These

instruments provide final separation of the pesticide residue from remaining components on a

column of absorbent via several different mechanisms. The instruments also provide identification

and a degree of confidence that the compounds eluting from the column at the same retention time as

standard materials are indeed the pesticide
206

residues. This is not always a certainty, however;

it would be impossible to test every variety of every

crop grown in every soil type and treated with every

herbicide, insecticide, and fungicide for interferences in the residue method.

For each of these two types of instrumentation,

there are numerous types of detectors. For instance,

the gas chromatography may be equipped with flame

ionization, electron capture, alkali-bead flame, photoionization, flame photometric, Hall electrolytic

conductivity detectors, or combinations thereof.

These detectors operate under different principles

and have the ability in some cases to detect only

certain classes of chemicals. Residue chemists use

this detector specificity to great advantage in

method development and residue analyses.

High-pressure liquid chromatography can have

ultraviolet absorption, fluorescence, photoionization, photodiode array, or electrochemical detectors. Some researchers have developed specific reactions that are employed on-line after the column

separation but prior to detection. This difficult type

of in-situ derivatization, regardless of whether

ultraviolet, visible absorption or fluorescent detection is used, is known as a post-column reaction

detector. Symptomatically, this points out the great

lengths the chemist is willing to go in order to

achieve selective and sensitive analytical methods

for the measurement of pesticide residues.

Emerging Technologies

Two of the emerging technologies for detecting

pesticide residues in foodstuffs are the mass spectrometer and the immunoassay. The mass spectrometer, whether it is coupled with the gas or liquid

chromatography, can provide a positive identification of a pesticide residue component by virtue of

its peculiar mass-fragmentation pattern. The specificity of the mass spectrometer is the real advantage, although for many compounds it also has great

sensitivity. For difficult to detect compounds, there

is always the option of derivatization, as with the

other quantitation techniques. The mass spectrometer can also utilize several different ionization

modes such as chemical ionization, electron impact,

field resorption, or fast atom bombardment.

It is feasible to use the Luke-acetone extraction

procedure (19) to isolate pesticide residues from the

crop matrix, provide a gross cleanup with gel

permeation chromatography (24, 25, 26) or florisil

column absorption, and then proceed to GC-MS for

detection. Coupling the resolving power of capillary chromatography with the specificity of GC-MS

would allow screening a large number of compounds

through its spectral library. Mass-fragmentation patterns matching particular compounds could be

reanalyzed by selective ion monitoring (SIM) for

confirmation and quantitation. By analogy, these

techniques are now being used to analyze approximately 165 compounds in water and sediment for

priority pollutants (26) Sensitivity of detection may

in some cases be a severe disadvantage of this technique. As described in 40CFR180, crop tolerances

in RACs vary widely by compound and crop type,

which could result in some samples in violation being undetected due to the differences in tolerance

levels of pesticides. For instance, one pesticide may

have a tolerance of 50 ppm in corn grain, and

another pesticide may have a tolerance of 0.05 ppm.

An analysis screen set for the high-level tolerance

would miss the low level and thus the sensitivity

of the screen must by approached with knowledge

of the tolerances. These tolerance levels could be

easily identified by tabular presentation of compounds and RACs.

The use of mass spectrometry as a tool for analysis of pesticide use has been dramatically increasing, although instrument size and expense are a

drawback. University and other small laboratories

may not be able to justify the expense of dedicating

a mass spectrometer for residue analysis. Contamination of the source with large amounts of chemicals from non-residue level use is an additional potential problem. Bench-top models with smaller

price tags are being developed and could ultimately

have significant impact for use as a multiresidue

screening tool. One area for vigilance is that some

classes of similar pesticides and degradants could

conceivably yield the same fragment ions.

The other area of emerging method technology

is the development of immunoassay for pesticide

analysis. Immunoassay are generally applicable to

pesticide chemistry, and these immunochemical

techniques are highly specific, sensitive, rapid, costeffective analytical methods (9,10). They owe their

great sensitivity and specificity to biological systems

that can produce the reagent antibodies that bind

with high affinity to compounds of interest. Immunoassay are very sophisticated and require a

certain proficiency to develop. Each intended use

of the immunoassay has to be carefully considered

prior to initiation of development efforts. Choice

of the hapten, preparation of the conjugate, generation of the antibodies, and incorporation of the antibodies into an assay all have to be carefully and

thoughtfully worked out prior to the very important demonstration of method viability by analysis

of samples (11).
207

The most frequently mentioned concern with immunoassay results is the nagging possibility of unsuspected interactions with unknown components

in the sample. This doubt is somewhat magnified

because of the absence of color development in positive results, which is the opposite of the traditional

color development in positive findings of derivatizational spectrophotometric methods. Thus, additionally colored solutions also create a concern (12).

Since nearly all pesticide immunoassay are competitive binding experiments, any interferences that

inhibit “complexation” of the antibody with the

tracer yield incorrect positive findings.

Pesticide residues of regulatory concern in foods

are often composed of mixtures of the parent and

degradates (i.e., total toxic residue). In such cases,

the extreme specificity of an immunoassay method

may actually be a handicap. Knowledge of the ability of the antibodies to recognize the degradates is

critical. In some cases, degradates could be substantially more sensitive than the parent. Therefore, a

positive result above the tolerance level in a multiresidue screen may also be a false positive. In some

literature studies, only 30 to 50 percent of the positive occurrences actually contained the analyte as

confirmed by GC/MS(l 1). Thus, use of immunoassay for multiresidue screening has to have the potential for confirmation by other analytical techniques and should be evaluated to determine if the

potential level of false positives is acceptable. In general, the immunoassay technique appears to offer

excellent opportunity for use as a multiresidue

method especially because of the low incidence of

false-negative detections. Using this technique, the

analyst can screen many more samples than previously possible with conventional techniques. However, more research directed toward field validation will be required to evaluate the immunoassay’s

reliability.

Multiresidue Technologies

The major advantage of a multiresidue method

for the analysis of pesticides is that the method allows the analyst the opportunity to look for the presence of many pesticides at once. EPA could further

encourage the development of these multiresidue

methods by focusing on development of enforcement methods, not on the total toxic residue but on

the identification and selection for analysis of the

most significant analyte of a pesticide whether it

be parent, metabolize, or degradate on a case-bycase basis. This would of course direct method development toward chemical classes or functionalities, as with the existing multiresidue methods, The

private sector, especially the food producers, food

processors, and contracting laboratories, would

benefit greatly from the ability to screen a raw agricultural commodity or processed food for multiple

pesticides.

Companies who are processing food for distribution are conducting quality-control analyses. The

National Food Producers Association (NFPA) uses

PAM 212-2 (Luke) acetone extraction procedure.

Four aliquots are taken to analyze for chlorinated

hydrocarbons, organophosphates, carbamates, and

substituted ureas. If they have special monitoring

interests, they revert to specific methodology (13).

Campbell Soup Company uses a similar ploy in

using the 212-2 extraction procedure and expanding the florisil-elution parameters to include a large

number of compounds. Analytical chromatographic

conditions are also expanded to include the analytes of interest (14). The Dried Fruit Association

in California operates under similar procedures (15).

The NFPA also conducts research on the effects of

pesticide residues in food processing (13).

Food processors generally conduct residue analyses (16,17) but they are not involved in methods

development. They are contractually requiring

pesticide-use history from growers to assure that

pesticide label requirements were followed. Classical methods are then used for additional quality

control (17).

The California League of Food Processors provides growers information on pesticides that may

legally be used in California, that is, the tolerance,

maximum-use rate, and the frequency of application for a particular crop. It also provides several

forms dealing with pesticide treatment and crop history including guarantee forms, report forms, notices to growers, and refusal forms (18).

Quality assurance and litigation samples lead the

analyst of a contract laboratory to the more general

or screening procedures. In these cases, qualitative

identification of the analyte can be as important as

quantitation. The analyst will rely on methods from

Federal regulating agencies such as EPA, FDA, and

USDA, or from peer-review organizations, like

AOAC or ASTM (19).

Actually, the regulating agencies are in the best

position to coordinate multiresidue method development, especially EPA, since they are in possession of all the pesticide registrants’ information on

the physical properties, product chemistry, metabolism data, and means of residue analysis.

The pesticide registrants are required to submit

an analytical method that is not designated as “com-
pany confidential” for enforcement purposes. Additionally, EPA is requiring registrants to report the

behavior of pesticides in the four FDA multiresidue

protocols (20). However, some judgment is needed

with respect to requiring the fit in these multiresidue methods for a parent molecule when the

parent has been demonstrated in radio-labeled crop

metabolism studies to rapidly and extensively

metabolize. Whether expending a great amount of

effort for multiresidue method development of pesticides with low toxicity (Category D or E) is worthwhile or not is also an issue to consider. Thus, the

starting point for decisions on multiresidue development is in the hands of the regulating agencies.

Federal agencies should upgrade their technical

approach to multiresidue technology. Industry

seems to have the opinion that these techniques are

antiquated, but in fact major gains can be made

through modernizing the analytical step. Registrants are required to validate residue methodology

used to develop the tolerance database by analysis

of endogenous residues from the radio-labeled metabolism studies. This validation certainly identifies the solvent system needed for extraction in the

enforcement method, as well as the potential

selected analyte for multiresidue methodology.

Standardization of chromatography materials with

respect to size, surface area, moisture and absorptivity, for instance, would be beneficial to the analyst and help reduce inter-laboratory variability.

Capillary and wide-bore capillary columns have become very practical in the laboratory since the advent of fused silica-bonded phase columns and they

offer substantial increase in resolution from packed

columns. Analytical detection systems in multiresidue methods are basically reduced to electron

capture and thermionic specific for bas chromatography and UV-VIS and fluorescence detection for

HPLC. Multiresidue technologies need to be expanded to include selective ion monitoring of mass

spectrometry, especially for those pesticides or

degradates that do not have a heteroatom to allow

selective identification.

Perhaps the most visible item to be improved is

presentation of the methodology in the Pesticide

Analytical Manual Volume I (PAM-I). A wealth of

information is harbored in its chapters if the analyst masters the system. Improvements could be

made by using clear block-letter headings describing procedures instead of relying on the numerical

codes. Methods should be presented in complete

form to allow a more concise flow of information,

as in the style of the AOAC Manual (8). References

to supplemental methods should be presented in the

appendix to PAM-I while listing pesticide and

cleanup procedures.

The most innovative step one could undertake

would be the computer indexing of the PAM data

base. Each pesticide entry file should have all the

chromatographic conditions, approved methods,

supplemental methods, and data on chemical structure. These data made available as public information in the form of a personal computer (PC) disk

would give the analyst easy access to the analytical

data. In those instances in which a class of compounds are to be analyzed, this would be a starting

point in determining universal conditions and the

development of multiresidue methods. The key to

acceptance is ease of use (21)0

An analyst could further narrow the scope of the

multiresidue investigation by consulting an information center or database consisting of agronomic

practices, crop registrations (22), pesticide fate, and

toxicological significance. For example, Dr. Phil

Kearney of the USDA-ARS developed a list of pesticides used on corn by consulting a half-dozen databases such as EPA information and state surveys

(23).

Similar use patterns could be developed for

vegetables, fruits, nuts, and other field crops based

on regional use patterns. From these data, optimal

selections of analytes would be made for the analyst. For example, the Arizona Department of Agriculture (ADA) requires certified applicators to register the compound being applied, the location of

the field to be sprayed, and the registration form

be filed with ADA. These data could also be compiled and made available to interested parties on

a PC disk for easy access.

If the Federal government wanted to stimulate

analytical method development, and particularly

multiresidue methodology in the private sector, two

things could be done to affect pesticide industrial

development. The first would be to expedite the

EPA review process. Currently, pesticide registrants are asked to internally review their petitions

for registration package completeness. Still the EPA

takes 12 to 18 months to respond to a submission.

Expeditious review, whether by additional staffing

or improvements in efficiency of the review process, could shorten the registration cycle.

The enforcement method seems to be the avenue

that the agencies could use to encourage the pesticide industry to enhance multiresidue technology.

Conditional registrations could be granted based on

the submission of scientifically credible crop-
209

residue studies with the proviso that the industry

chemists attempt to fit the enforcement methods to

the multiresidue scheme. Since it takes a while for

a product to achieve widespread distribution and

a “dent” in the marketplace, by this time the multiresidue enforcement method could be in place for

screening purposes.

The pressure on the industrial chemist and the

pesticide industry in general could be somewhat

relieved by prolongation of the patent life to partially compensate for the 5 to 7 years spent in regulatory clearance studies. Since multiresidue method

research is complex, time-consuming, and expensive, allowing industrial chemists greater freedom

without delays in the pesticide registration could

produce the desired results. Prompt evaluation of

registration data would result in a longer market

life, more profitability, and a propensity on the part

of industry to provide resources toward developing methods for surveillance. In other words, if EPA

reviews were expedited and patent-life prolonged,

industry would not object to additional methodology requirements. Other options require the higher

risk, and less advantageous, pouring of funds into

long-term contracts or grants.

Governmental agencies should combine their resources to provide analytical pesticide residue training, similar to the Advanced Pesticide Residue Analysis workshop jointly sponsored by the US EPA

National Enforcement Investigations Center and

the New Jersey Department of Environmental Protection. The concept of this workshop could be enlarged to include analysts from the private sector.

Recent demands in the analytical support of registration studies have resulted in qualified personnel

being the limiting factor in laboratory expansion.

Training sessions that reviewed the techniques of

the PAM-I would be of value to the method development chemist as he researches for a sensitive and

selective analytical scheme. Education of the

researcher developing methods that coincide with

EPA and FDA requirements would go a long way

in attaining these goals.

Conclusions

The organization and direction of multiresidue

methodology seem to rest with the EPA and FDA.

The pesticide registration database complete with

metabolic or degradate information is known by the

EPA, and the pesticide monitoring techniques are

known by the FDA. Incentives for development of

these methods, whether in the form of appropriations, grants, or conditional registration, should be

fostered by these agencies.

Industry should be requested to develop enforcement methods focused on analytes that are most

likely to be present based on compound half-life calculations and the metabolic degradation pathway.

FDA is facing an impossible task when asked to

screen a crop for a few hundred compounds, as well

as all their degradation products. It would be preferable to select one or two representative moieties as

a biomarker to be incorporated into a multiresidue

screen. If residues and the incidence of violation

warrant further analyses, the total residue method

should be used as supplied by the registrant.

The applicability of enzyme inhibition and immunoassay should be evaluated for pesticide residue analyses. Federal appropriations should be used

to evaluate the number of classes of compounds that

can be screened by these techniques. How broad

are their applications with respect to sample matrix? Classical methods should be used simultaneously with the screening techniques to validate their

accuracy. If these questions and conditions are satisfied, EPA could further promote promising techniques such as immunoassay, by acceptance as enforcement methodologies,

Multiresidue techniques are important and can

be improved through a concerted effort. Revision

of the PAM-I format, a PC disk database access, and

upgraded analytical procedures including capillary

chromatography and GC-MS-SIM would encourage

this development.

Agency and private sector meetings or training

sessions should be promoted to advance discussions

for solutions to problems. Ultimately, the greatest

step in solving the technical problems of pesticide

residue analyses will be the enhancement of communication by all parties involved in the agricultural arena.

1.

2,

3.

4.

5.

6,

7.

References

Pollack, Robert, Analytical Development Corporation, Colorado Springs, CO, personal communication, January 1988,

Craven, Don, Craven Laboratories, Austin, TX,

personal communication, January 1988.

Ganz, Charles, ENCRS Laboratory, WinstonSalem, NC, personal communication, January

1988.

Hughes, Don, Hazleton Laboratories, Madison,

WI, personal communication, January 1988.

Maliami, Nancy, Morse Laboratory, Sacramento,

CA, personal communication, January 1988.

Spittler, Terry, Cornell University, Geneva, NY,

personal communication, January 1988.

U.S. Environmental Protection Agency; Pesti-
210

8.

9.

10.

11.

12.

13.

14.

15,

16.

17.

18.

cide Assessment Guidelines, Subdivision O:

Residue Chemistry, October 1982; NTIS PB83-

153981.

Association of Official Analytical Chemists,

Officia) Methods of Analysis, S. Williams (cd.),

AOAC (14th edition), 1984.

Hammock, Bruce, University of California,

Davis, CA, personal communication, January

1988.

Hammock, B. D., Mumma, R. O., “Potential of

Immunochemical Technology for Pesticide

Analysis,” In: Pesticide Analytical A4ethodo]-

ogy, J. Harvey, G. Zweig(eds.), ACS Symposium

Series Number 136 (Washington, DC: American

Chemical Society, 1979).

Wratten, S.J. and Feng, P. C. C., “Pesticide Analysis by Immunoassay,” In: Development and

Applications of Immunoassay for Food Analysis J.H. Rittenburg (cd.) (Elsevier, 1988).

Ferguson, Bruce, ImmunoSystems Inc., Biddeford, ME, personal communication, January

1988.

Elkins, Ed, National Food Processors Association, Washington, DC, personal communication,

January 1988.

Lento, Harry, Campbell Soup Co., Camden, NJ,

personal communication, January 1988.

Steffen, Ed, Dried Fruit Association, Fresno,

CA, personal communication, January 1988.

Smalligan, Wayne, Gerber’s Baby Food, Fremont,

MI, personal communication, January 1988.

Vetro, Robert, Delmonte Foods, Walnut Creek,

CA, personal communication, January 1988.

California League of Food Processors, Pesticide

Lists and Program Forms, 1112 “I” Street, Suite

100, Sacramento, CA 95814.

19.

20.

21.

22.

23.

24.

25.

26.

U.S. Food and Drug Administration, FDA Pesticide Analytical Manual—Volumes I, June 1982;

NTIS PB82-911899.

U.S. Environmental Protection Agency, Pesticide Assessment Guidelines, Subdivision O—

Addendum: Residue Chemistry Data Requirements for Analytical Methods in 40 CFR Part

158.125—Muhiresidue Protocols, May 1986;

NTIS PB86-203734.

Williams, Michael, ABC Laboratories, Columbia, MO, personal communication, January

1988.

Crop Protection Chemicals Reference, Joint venture of Chemical and Pharmaceutical Press and

John Wiley & Sons, (2nd Edition) 1986.

Kearney, P. C., USDA-ARS, Beltsville, MD,

“Current Status of Herbicides in Groundwater,”

presented at the 28th Weed Society Meeting,

February 1988.

Association of Official Analytical Chemists,

“Organochlorine Pesticide Residues in Poultry

Fat, Gel Permeation Chromatogaraphic Method,

First Action,” In: Official Methods of Analysis,

S. Williams (cd), AOAC, (14th edition) Section

29.037-.043, 1984.

Hopper, M., “Automated Gel Permeation System for Rapid Separation of Industrial Chemicals and Organophosphate and Chlorinated Pesticides from Fats,” J. Agric. Food Chem.,

Nov/Dee: 1038-1041, 1982.

Method 3640, “Gel Permeation Cleanup,” Test

Methods for Evaluating Solid Waste, Physical/Chemical Methods, Laboratory Manual, SW846-USEPA.
211

Pesticide Residue Monitoring in Canada

W. Harvey Newsome, Bureau of Chemical Safety, Foods Directorate, Health and Welfare

Canada

and

Geraldine F. Graham, Bureau of Field Operations, Field Operations Directorate, Health

and Welfare Canada

Contents

Page

Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , , ,211

Pesticide Residue Monitoring in Canada . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , ,212

Programs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0 . . . . . . . . . . . . . . ........0 .. ...212

Sampling Strategy for National Surveillance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..213

Current Analytical Methodology. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ........213

Research and Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ............213

TLC-Enzyme Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...,214

Common Fragment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ................,214

Immunochemical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..............214

Robotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...215

Appendix 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..........215

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...216

Figure

Figure Page

1. Monitoring Programs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..........212

Table

Table Page

l. Characteristics of Immunochemical Methods Developed at Health Protection

Branch . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..215

Abstract

The Health Protection Branch of the Department

of National Health and Welfare establishes maximum residue limits for pesticides in food in Canada, and it is responsible for ensuring that commodities offered for sale comply with these limits

through surveillance and compliance programs. In

addition, data are gathered on the levels of residue

in a variety of foods to determine the dietary exposure of the population to these chemicals.

Approximately 3600 samples are analyzed annually through four programs designated as surveillance, compliance, data gathering, and total diet.

The surveillance program is divided into regional

and national components; the former generates

data on residues and foods that are of local concern

while the national component determines the state

of compliance of selected foods in the marketplace

across the country. The compliance program investigates and solves problems identified in the surveillance project. The data gathering program conducts analyses of a specialized nature in response

to concerns arising from new toxicity data or gaps

in the residue database. The total diet study provides data on the actual dietary intake of pesticides

from foods prepared as for consumption,

Methodology used for monitoring relies heavily

upon a multiresidue procedure capable of determining 155 compounds. An additional 37 compounds predetermined by specific methods. Current research is directed toward increasing the

efficiency and scope of monitoring methods using

immunochemical, degradation to a common fragment and robotic approaches. The characteristics

and applicability of these methods are discussed.
212

Pesticide Residue Monitoring in

Canada

Monitoring for pesticide residues in food is conducted at the federal level by the departments of

Agriculture, Fisheries and Oceans, Canadian Grain

Commission, National Health and Welfare, and in

the provinces by the laboratories of the ministries

of Agriculture and Food. The objectives of the various projects differ substantially—from providing assurances to farmers that pesticides used according

to label directions will not result in residue problems and approving shipments for export to enforcing compliance with maximum residue limits (MRLs)

established under the Food and Drugs Act. The

Health Protection Branch of the Department of National Health and Welfare establishes the MRLs and

is responsible for their enforcement through surveillance and compliance-type projects carried out

in five regional laboratories situated across Canada.

In addition, data are collected on the occurrence

of residues in order to determine the dietary intake

and to ensure that the lowest possible exposure consistent with effective pest control is attained.

Programs

Monitoring programs conducted by the Branch

are divided into four categories: data gathering, total diet, surveillance, and compliance. A summary

of these and an indication of the proportion of samples directed to each is given in figure 1.

The objectives of the Canadian program are very

similar to those described for the United States (19)

and the types of program also resemble each other

closely, although the numbers of samples analyzed

in Canada are smaller than those outlined for the

United States (8). Thus, the total diet, data gathering, and regional surveillance projects in Canada

are comparable to the total diet, selected survey, and

district option in the United States. However, Canadian national surveillance and compliance projects

do not have U.S. equivalents. Another major difference between the two countries’ approach is that

Canada does not have a separate program for imports but includes these items in most surveys in

proportion to consumption.

Data gathering projects are designed to collect information on the occurrence of specific pesticides

in the food supply, and they often involve biased

sampling. Specific methodology, as compared to

multiresidue methods, is often required, and projects are initiated as a result of new information on

the probable occurrence of compounds or their

Figure 1 .—Monitoring Programs

TOTAL SAMPLES-3585

National Regional Data Total

surveillance surveillance Compliance gathering diet

(1100) (525) (525) (900) (535)

1/1 dom, / 4/1 dom. / 1/2 dom. / national 224

import import import composites

metabolizes or in response to concerns arising from

new toxicity data. Approximately 900 samples are

analyzed annually in this program. The total diet

program is similar to that conducted in the United

States and, in its present form involves the preparation of 161 food items as consumed. One hundred and twelve composites of these items are then

made, representing 99 percent of the Canadian diet.

Samplings are conducted twice annually from

different cities representing the five regions across

Canada, such that all regions are covered in a two

and a half year cycle. All composites are analyzed

by the multiresidue method (9), and six additional

compounds are determined by specific methods.

The resulting data reflect actual pesticide intake that

remains after trimming, washing, and cooking of

foods.

The surveillance program consists of national and

regional components. The national surveillance

component is designed to determine the state of

compliance of selected food commodities in the

marketplace with respect to selected pesticides. Approximately 1,100 samples are analyzed annually,

approximately one half of them by multiresidue

methodology. A smaller number of samples (525)

is analyzed in the regional surveillance project. Regional surveillance is planned separately by inspection and laboratory staff in each region and is designed to emphasize commodities and pesticides

that are of local, rather than national, importance.

This program uses information gathered on local

pesticide usage, infestation problems, and crop conditions. Commodities are included where pesticide

misuse is suspected.

Data obtained from surveillance projects are compiled each year, but are not published or stored in

a computerized database. They are used internally

to direct future surveillance and compliance

projects and are available on request to other interested parties, including international agencies such

as WHO/FAO.

The national surveillance component has only

been in place in its present form since 1985. Eleven

out of fourteen commodities tested have shown a

state of compliance of at least 99 percent. The three
213

exceptions are now included in the compliance

project.

The compliance program is designed to investigate and solve residue problems identified by the

national or regional surveillance components. This

project is instrumental in preventing the sale of

foods containing residues in excess of the MRL. Historically, a wide variety of pesticides have been

found at violative levels. For example, in 1986/87,

thirty-one different compounds were involved, with

fungicides and organophosphates being the most

numerous. Because the use of pesticides that are

registered in other countries but not in Canada often

results in residues that exceed our MRLs, residue

problems have occurred more frequently with imported products than with domestic ones. Therefore, compliance efforts are concentrated on importers, who, under Canadian law, are responsible

for ensuring the products they import comply with

the Food and Drugs Act. Actions taken if violative

residues occur range from refusal of entry to prosecution of importers who repeatedly import violative products.

Sampling Strategy for National

SurveiIlance

The food supply is divided into 14 commodity

classes such as vegetables, meat, dairy products, and

fruit. These classes are ranked according to consumption, pesticide application, potential for residues, and data available from other agencies. Of

these classes, fruit and vegetables represent approximately 35 percent of the Canadian diet and have

the highest potential for residues. Thus, they are

designated as constituting a minimum of two thirds

of the surveillance samples. Thirty-five items from

these commodity classes, representing 90 percent

of the apparent consumption are selected for analysis once over a 10 year period. The highest consumption items representing 50 percent of the diet

are analyzed twice over the 10 year period. For each

pesticide-commodity combination, 100 samples are

analyzed, so that a 4.8 percent violation rate would

be detected with 95 percent confidence (2). These

samples are procured in proportion to the geographic distribution of their origin, i.e., by province

of production for domestic commodities and by

country of origin for imports.

In Canada, 235 pesticides are registered for use

on food. Those recommended in provincial spray

calendars are considered to represent those in actual use and are ranked using such factors as volume of use, persistence, and toxicity. The selection

of pesticides to be determined on imported foods

is based upon the existence of a tolerance in the

country of origin and weighted by the frequency

of occurrence of previously detected residues. The

FDA surveillance index (20) is also heavily relied

upon to furnish a criterion for priority, Thus, a list

of commodity-pesticide combinations is constructed.

For those compounds that can be determined by

multiresidue methods, pesticides can be included

down to a low level of priority, while those requiring specific methods must be selected from the high

priority portion of the list.

Current Analytical Methodology

As indicated previously, a multiresidue method

(9) is used for surveillance wherever possible. This

procedure, which is capable of determining 155

compounds in a variety of fatty and non-fatty foods,

involves cleanup of an acetone extract by automated

gel permeation chromatography followed by determination by capillary gas-liquid chromatography

using at least two detectors—usually a Hall detector in the halogen mode and a thermionic nitrogen/phosphorous detector. HPLC with post-column

derivatization as described by Krause (5) is used for

methyl carbamate pesticides. An additional chromatographic cleanup on Florisil is required with

some commodities.

Specific methods are used for an additional 37

compounds that cannot be determined by the multiresidue procedure. These compounds are examined

as the need arises and, on average, each consumes

the same resources as the multiresidue procedure.

Often, members of this group of pesticides are insufficiently volatile for gas chromatography and do

not contain chromophoric groups necessary for detection after HPLC. Examples are the ethylenebis(dithiocarbamates) fungicides and daminozide.

Others such as maleic hydrazide, glyphosate, diquat, paraquat, and the organotin compounds cyhexatin and fentin require unique cleanup steps due

to their polar nature. A complete list of these compounds is given in appendix 1.

Research and Development

In an effort to improve the efficiency of the monitoring process, as well as to identify new compounds such as metabolizes and degradation products, research is conducted in the Health Protection

Branch into the development of analytical methods.

The private sector does not conduct method development as such but relies on methods published in
214

the scientific literature or in manuals developed by

Federal departments. The methods developed by

the branch may classified as four types and will be

discussed individually as: 1) TLC-enzyme inhibition, 2) degradation to a common fragment, 3)

immunological, and 4) robotics.

TLC-Enzyme Inhibition

Two rapid screening techniques, qualitative in nature, have been studied in our laboratory to determine rapidly, and with as little sample purification

as possible, whether a sample contained violative

residues. Both rely on separation of the pesticides

by thin-layer chromatography, followed by treatment of the developed plate with an enzyme or enzyme system and substrate. The first of these methods has been reviewed by Mendoza (10) and consists

of detection of carbamate and organophosphate insecticides with an esterase preparation followed by

a chromogenic substrate. Zones of inhibition indicated the presence of cholinesterase inhibitors, the

identity of which could be indicated by the Rf

. Several factors affected the sensitivity of the assay,

including source of enzyme, substrate, and pretreatment of the plate with an oxidant to convert thiophosphates to their oxygen analogues. The disadvantage of the method, which prevented its routine use,

was the presence of a number of naturally-occurring

inhibitors in food extracts.

A more promising development is that of TLCphotosynthesis inhibition, which is applicable to

those herbicides that inhibit photosynthesis such

as phenylureas, phenyl carbamates, and triazines

(6). After chromatographic separation, the plate is

sprayed with a suspension of chloroplasts, followed

by the redox indicator 2,6-dichloroindophenol and

exposure to light. Photosynthetic inhibitors appear

as blue spots of unreduced dye. The method requires

little sample workup other than initial sample extraction and partitioning into dichloromethane, and

it is relatively immune to interference. Detection

limits are satisfactory for compliance purposes.

Common Fragment

This approach involves the conversion of pesticides with different properties, such as polarity and

vapor pressure, to a common entity, permitting the

determination of several compounds in a single

analysis. The technique has been used for the detection of phenyl urea herbicides in a variety of sample types (4), and it provides information as to the

total burden of a class of compounds in a food. This

concept may also be used to determine alachlor,

diethatyl, and their 3,5-dichloroaniline-containing

metabolizes by hydrolysis to 3,5-dichloroaniline and

determination of 3,5-dichloroaniline by GLC (15).

Similarly, iprodione, vinclozolin, and procymidone

are also determined by alkaline degradation to 3,5-

dichloroaniline (18). The obvious disadvantage of

this approach is that if excessive residues are encountered, individual determinations must be made

to identify the offending compound,

Immunochemical Methods

The widespread successful use of immunoassay

techniques (3) in clinical laboratories prompted us

to evaluate its applicability to pesticide residues in

foods. Both radioimmunoassay (RIA) and enzymelinked immunosorbent assays (ELISA) have been

developed for compounds ranging from the nonpolar polychlorinated biphenyls (11) to the watersoluble fungicide carbendazim (12, 15). Either immunochemical approach resulted in methods that

correlated well with conventional chemical analyses for all compounds we have studied. The nonpolar PCBs posed the greatest problem, requiring

sample purification as extensive as that required

for gas chromatography if false-negative data were

to be avoided. In contrast, carbendazim could be

determined in crude ethyl acetate extracts without

prior cleanup and was not subject to interferences.

Similarly, methods for the fungicides metalaxyl (13),

iprodione (16), and triadimefon (14) did not require

any sample preparation other than initial extraction.

The specificity of immunochemical methods is

generally sufficient for screening purposes but, as

the data summary in table 1 indicates, varies greatly

with the assay and is probably a reflection of the

structure of the pesticide. For example, the selectivity of the assay for thiabendazole is very high,

with low cross-reactivity for related compounds

such as 2-benzimidazoleurea or carbendazim. In

contrast, vinclozolin and procymidone react with

antibody directed toward iprodione to a higher degree than does iprodione itself. Similarly, the herbicides metolachlor and diethatyl have considerable

cross-reactivity with metalaxyl antiserum, further

emphasizing the screening nature of the analysis.

The merit of ELISA compared with RIA lies in

the relative safety and availability of reagents and

in the simplicity of associated counting equipment.

However, RIA is often more rapid, requiring fewer

incubation steps and produces steeper inhibition

curves, which result in greater sensitivity, A larger

number of samples may be processed at one time

with ELISA, resulting in low unit cost. Compared

with conventional specific analyses, ELISA is ca-
215

Table 1 .—Characteristics of Immunochemical Methods Developed at

Health Protection Branch

Compound Assay Quantitation Major Cross Sample

determined type limit (ppb) reactions

Aroclor 1260 RIA 2 Aroclor 1254 milk

Carbendazim RIA 50 2-benzimidazole cucumber

urea

Metalaxyl ELISA 100 metolachlor, tomato

diethatyl

Thiabendazole ELISA 30 nil potato

Carbendazim ELlSA 350 2-benzimidazole apple

urea

Iprodione ELISA 100 vinclozolin, tomato

procymidone

Triadimefon ELISA 500 triadimenol apple

pable of producing four to five times the number

of determinations per day.

Robotics

Robotics is being studied as a means of reducing

the labor-intensive component of conventional multiresidue analyses. Two implementations of this

technology are being evaluated—one that carries out

the liquid-liquid partition step in the Luke et al. procedure (7), and another that prepares milk samples

for the determination of a number of organochlorine compounds by gas-liquid chromatography.

The system used for partitioning (1) consists of

a Cyberfluor Labotix robot arm, stirrer, and liquid

handling apparatus under control of a microcomputer. Aliquots of sample extract are manually added to a flask where partitioning is carried out by

a series of stirring actions with dichloromethane.

After phase separation, the dichloromethane is recovered by the robot for concentration prior to

cleanup. Recoveries of standards added to several

commodities were comparable to those obtained

with the manual partitioning procedure.

For milk analysis, the entire extraction and cleanup

procedure was automated using a Zymark Corp.

arm and custom-built series of workstations. This

apparatus permits the weighing of sample, extraction with organic solvent, centrifugation, column

chromatography, and collection and evaporation of

three fractions prior to gas chromatographic analysis. Thirty-two compounds, in addition to PCBs

are determined using an autosampler and data acquisition system. When evaluated against manual

sample preparation, the robot was found capable

of doubling the weekly output. The coefficient of

variation at the 1 ppb level was 15 percent for the

automated system compared with 8 percent manually. Accuracy was equivalent for both systems.

Method development is now being conducted into

the further application of immunochemical and robotic procedures, as well as such techniques as solid

phase extraction for inclusion in the multiresidue

method. In addition, new pesticides are continually being tested for inclusion into the existing multiresidue method. Since the analytical problems

encountered in monitoring the food supply are common to both the United States and Canada, both

countries benefit from new developments arising

from research in North America or abroad. An excellent mechanism for communication and exchange

of this technology is the Association of Official Analytical Chemists. This international organization of

scientists from government, industry, and academia disseminates new findings at its annual meetings and through publication in a journal. In addition to being a forum for discussion of new

approaches, methods are validated through a process of collaborative study in several laboratories.

Appendix 1

Pesticides Determined by Single Residue Methods: aldicarb, amitraz, *benomyl, bentazon, biphenyl,

*chlorophenols, “daminozide, desmedipham, dichlone, diquat, *diuron, dodine, *EBDC, *ethylene

dibromide, ethephon, ethylene thiourea, fluazifopbutyl, glyphosate, imazalil, iprodione metabolizes,

*maleic hydrazide, methiocarb, methomyl, *methyl

bromide, napthalene acetic acid, naptalam, “organotin

compounds, oxamyl, oxydemeton-methyl, “paraquat,

o-phenyl phenol, pyrethrins, terbutylazine, thiabendazole, triallate, triforine, vinclozolin metabolizes
216

Those compounds marked with * have been identified by GAO as needing single residue methods.

References

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1980.

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Brinkman, Th.,U.A., Intern. ]. Environ. Anal.

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8. McMahon, B.M. and Burke, J. A., J. Assoc. Off.

Anal. Chem. 70:1072, 1987.

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Newsome, W.H. and Shields, J. B., J. Agric. Food

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Anal. Chem. 70:1025, 1987.

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217

Pesticide Monitoring Program in Mexico

Silvia Canseco Gonzalez, Animal and Plan Health Office, Agriculture Department Mexico

The main objective of the Animal and Plant Health

Law of the Mexican Republic is to protect animals

and plants from pests and diseases. In addition, the

law provides the Secretary of Agriculture and Water

Resources the facilities to exercise control over the

quality of biological and chemical products applied

to animals and vegetables as well as to prevent

agrarian activities from originating health risks and

environmental contamination. This is carried out

through the Plant and Animal Health General Direction, responsible for pesticide registration and control, This office takes care of setting tolerances and

checking the quality of the formulations available

for the growers.

The system to control these chemical compounds

in Mexico involves separate aspects; the law requires the registration of import, manufacturing,

development, and distribution firms. It maybe considered that great advances have been made in the

regulation of these firms in the past 18 months.

Equally, the registration of compounds sold in

Mexico has kept a very acceptable level, as well as

the registration of technicians who supervise the

quality control in factories and who are responsible for usage recommendations in their own firms.

Pesticides, companies, and consultants are registered in the main offices in Mexico City; number

registration of sales and distribution is done through

the Agriculture Department officer in the Mexican

states. In 1974, the construction of a network of laboratories was begun to bring about quality control

of product formulas as well as to determine residue levels in affected crops. There are now 12 regional laboratories for pesticide analysis and one

central reference laboratory for pesticide residues

analysis of animal products. Five of these laboratories are able to conduct residue analysis as well,

and the Vegetables Growers Union has built a laboratory for the same purpose.

Some colleges and universities in Mexico are

making efforts to develop analytical methods, but

the task is centered most often upon the pesticide

industry and in the official laboratories. In both

cases, it may be said that more important than the

development of new methods is the implementation and verification of those methods developed

by benchmarks or published in the literature.

Some efforts have been made with respect to pesticide residue analysis with the objective of modifying some methods to make them more economical, but no conclusive results have yet been reached.

This year, the program involves the analysis of

2,200 samples of vegetal origin, specifically of the

following crops: chili peppers, green peppers, tomatoes, tomitillos, and strawberries. This is done using

the FDA Pesticide Analytical Manual procedures

already discussed and those modifications applicable to the country conditions.

The selection of the products to use against a pest

problem should be made on the basis of the manual

of Authorized Pesticides, which SAPAF edits and

reviews each year and which lists those compounds

that have complied with the requirements specified

by the law. Also it includes information about pests,

crops, and dosages that may be applied, safe intervals of application, and the residue limit that should

be observed.

Let’s use PAM procedures because the United

States is the main consumer of our agriculture exports, thus we check both the domestic and foreign

consumption.

However, during past years, economic factors

have had the following negative effects on our work:

—lack of proper maintenance of the equipment

—no new equipment

—loss of training technicians and inability to contract replacement personnel.

This situation is aggravated by the problems of

inflation and daily devaluation of the Mexican currency. This is reflected in the number of analyses

that can be carried out, reducing it a considerable

degree each year and thereby reducing the established capacity that the Secretarial de Agricultural

y Proteci–opecuaria y Forestal (SAPAF) once had.

During 1987, residue analyses were carried out on

970 samples of fresh agricultural produce. Since the

tasks of analysis is very specialized and practiced

by a small number of technicians, there is no interest in the reagent and solvent industry in Mexico

in maintaining a quality product that satisfies the

requirements of these analyses. For these reasons,

the reagents and solvents are imported. In addition,

much of the equipment and glassware is imported,

so the prices for these materials have increased.

Considering that the main problem with pesticides is misuse, we are implementing the use of a

prescription for selling pesticides. This means that

in order to purchase a pesticide, the grower must
———

218

present a paper written by a registered professional

indicating the crop, the pest, product, and dosage.

As I mentioned before, residue analysis is carried

out with the U.S. methodology whenever possible,

but we don’t eliminate the possibility of using those

recommended methods from international agencies

such as Codex Committee on Pesticide Residues.

We recognize the importance of fruit and vegetable trade between Mexico and the United States,

and we are very concerned about it. To this end,

we have instituted and analytical quality-control

program between FDA and my office to assure that

the pesticide residues in commodities involved in

trade are below tolerance levels.

Nowadays the Agriculture Department has the

tendency to implement pest control programs that

are part of an integrated pest management program

that that takes advantage of pests’ natural enemies;

and includes extending the use of old technologies

such as biological control. Last year we had spectacular results on the soybean crop, reducing the

use of pesticides by more than 50 percent.

Another technique is the use of sterile insects in

the campaign against the Medfly and the cattle

screwworm. Other pest control methods have been

developed that originated from the EDB ban:

hydrothermic treatment for the elimination of larvae in mangoes for export and the use of low temperatures as an agricultural sanitation treatment in

citrus exportation.

The biological pesticides Bacillus thuringiensis

is widely used for forestry pests in ecologically protected areas; we are conducting investigations on

the use of fungi against soil pests.

Despite all these efforts, it is recognized that pesticide use will continue to be the extensively used

means of pest control. Actions are being directed

toward making adequate use of pesticides in which

different government agencies, as well as the pesticide industry and professional organizations, participate by means of campaigns, qualification

courses, refresher workshops, and symposia, all oriented toward keeping pesticides a useful tool without excessive risk.

Strong efforts are being made to get economic

support from international agencies in order to let

us continue this task.

Having the opportunity to participate in events

such as this undertaking and others in which new

technologies are expounded and revised, or at least

having access to the information generated, can

help Mexico maintain an acceptable level of technological development in this area. Visitation by

specialists who might, upon observing conditions

in the nation, be in a position to offer a more practical and acceptable assessment, would be equally

useful. Nevertheless, the main problem is, and

according to our perspective will continue to be,

the lack of economic resources that permit us to

incorporate innovations in day-to-day work.

It is necessary to identify sources of financing to

continue the program and the technical assessment

that allow research into other analytical possibilities,
219

Developing Pesticide Analytical Methods for Food:

Considerations for Federal Policy Formulation

Sarah E. Taylor, Analyst in Life Sciences, Science Policy Research Division, Corgressional

Research Service

Abstract . . . . . . . . . . . . . . . . . . . . . . . .

Introduction . . . . . . . . . . . . . . . . . . . .

Federal Pesticide Residue Programs

Environmental Protection Agency . .

Federal Monitoring Program . . . . . .

Food and Drug Administration. . .

contents

Page

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..,,,., 219

and Analytical Methods. ..., . . . . . . . . . . . . . . .220

. . . . . . . . . . . . . . . . . . . . ,,,,... . . . . . . . . . . . . . . 220

,.,... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220

. . . . . . . . . . . . . .....$, . . . . . . . . . . . . . . . . . . . . . 221

U.S. Department of Agriculture/Food Safety and Inspection Service . . . . . . . . ....221

Federal pesticide Monitoring Program Evaluations . ...........................222

Analytical Technology and Program Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ....224

Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .......226

Questions to Consider as a Research Strategy is Defined . ......................226

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ...227

Abstract

Technologies available to detect, identify and

quantify pesticide residues in food have played a

key role in defining the structure and effectiveness

of Federal pesticide monitoring programs. Monitoring programs are used to assess public exposure

to pesticides and as a basis for enforcing pesticide

laws by detecting residue violations. Because of the

volume of samples that must reanalyzed and the

dearth of information on the pesticide treatment history of food samples collected for analysis, Federal

monitoring programs have been designed around

the use of multiresidue methods. However, multiresidue methods are unable to detect all residues

of interest, including a number of pesticides of high

priority because of their widespread use and toxicity levels. This gap in detectable residues has served

as a point of departure in recent policy debates concerning the appropriate direction of research in

methods development.

This report examines Federal pesticide monitoring programs and how analytical technology has

shaped them. It also considers program limitations

that have been identified in recent policy studies,

and raises questions about the role of analytical

technology in addressing these needs. This report

concludes that new analytical technology may offer an opportunity to address not only the gap in

detectable residues, but to help achieve even more

fundamental improvements in pesticide monitoring programs.

Introduction

Public health policymakers have long been concerned about the health implications of dietary exposure to pesticide residues. They have considered

analytical methods capable of detecting and quantifying pesticide residues basic to any program designed to control such exposure. Even before a Federal pesticide monitoring program was developed

under modern pesticide laws, this need for methods was recognized. For example, Dr. John Kerfoot

Haywood, head of the Federal Insecticide and Agricultural Water Laboratory in 1905, was disturbed

about possible health effects of pesticides and stated,

“[i]t is essential that these [pesticide] compounds

be analyzed by exact. . . uniform methods. . . .“(l).

During the past 3 years, several critical evaluations of Federal monitoring programs directed at

pesticide residues in food have advised that improved analytical methods were needed to enhance

the effectiveness of the programs. These recommendations have helped to focus public and congressional attention on analytical methods and have

fueled the reevaluation of Federal policies currently

underway inside the Federal agencies and by the

Congress.

However, the interest of the public and of policymakers in analytical methods has been borne of a

larger concern—that the government has been unable to supply to the public sufficient data to allay

concerns about the safety of pesticide residues in

the food supply. Some consumers have construed
220

the gaps in residue data as indicative of a grave and

unknown risk to public health (2). Some policymakers are desirous of more pesticide residue data

because they believe it will more clearly show that

the food supply is safe, and will help to restore public confidence in the effectiveness of the programs

already in place (3).

This report examines Federal pesticide monitoring programs and the relationship between analytical technologies and the design and limitations of

current monitoring programs. It considers recent

recommendations regarding program needs to improve the analytical methods used in the programs.

It also raises questions regarding how the goals and

design of pesticide programs in the future may influence the analytical methods policies developed

and implemented today.

federal Pesticide Residue Programs

and Analytical Methods

Pesticide analytical methods are scientific techniques used to detect, identify, and quantify pesticide residues. The technology generally used for

pesticide detection is gas-liquid chromatography

(GLC) and high-pressure chromatography (HPLC)

(4, 5). There are two general types of pesticide analytical methods in use today: multiresidue and single residue methods. Multiresidue methods are capable of detecting a number of pesticides having

similar chemical and physical properties in a test

of a single sample. Single residue methods are capable of identifying only one pesticide residue in

a food sample. In general, multiresidue and single

residue methods require comparable time and resources to conduct per sample. Therefore, multiresidue methods are considered more time and

resource efficient than single residue methods (6).

The advantages of multiresidue methods over single residue methods have helped to make them the

basis of current pesticide monitoring programs. In

addition, multiresidue methods capable of detecting large numbers of residues are useful in testing

samples when reliable information about the pesticides used on the commodity is lacking.

The Federal pesticide program is actually a patchwork of programs administered by the Environmental Protection Agency (EPA), the Food and Drug

Administration (FDA), and the U.S. Department of

Agriculture (USDA). Analytical methods play a key

role in the programs of each agency.

Environmontal Protection Agency

The EPA has central authority for the regulation

of pesticides under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA). Under the act,

EPA is required to grant a license (registration) for

pesticide chemical uses for which the applicant

(registrant] has demonstrated, among other things,

that “when used in accordance with widespread

and commonly recognized practice it will not generally cause unreasonable adverse effects on the

environment” (7). In determining whether a pesticide can be registered, among the factors EPA considers is whether residues that result from pesticide

use pose a dietary hazard to humans or animals.

For pesticides that will be used on a crop that will

serve as human or animal food, EPA is required to

grant a tolerance level for residues under the Federal Food, Drug and Cosmetic Act (FDCA). A tolerance level defines the maximum amount of pesticide residues that may remain in food (8).

A condition of registration for any pesticide for

which a tolerance is granted is that the registrant

supply to EPA a pesticide analytical method(s) capable of detecting and quantifying the active ingredient and the products of its degradation (9). An

EPA laboratory tests the method (validation) to assess the validity and reliability of the method (10).

The goal of the requirement is that the registrant

supply to the agency the analytical means to enforce

the pesticide tolerance. However, the requirement

has been ineffective in achieving this goal largely

because the methods supplied are often not practicable for regulatory purposes. Registrants generally fulfill the requirement for a method by supplying a single residue method. Because of the

limitations of single residue methods when compared with multiresidue methods, as noted above,

they are not feasible for routine use in monitoring

programs, except in limited circumstances where

some reliable information is available on the pesticide treatment history of the sample.

Federal Monitoring Program

Once a pesticide has been approved, the responsibility for monitoring residues in foods belongs to

FDA and USDA. FDA is responsible for monitoring all domestic and imported foods for pesticide

residues except for meat and poultry, which are

monitored by USDA. FDA and USDA enforce the
221

pesticide tolerances established by EPA. Foods containing illegal pesticide residues are considered

“adulterated.” FDA and USDA each have authority to inspect food to determine if it is adulterated

and to prosecute those who are involved in interstate commerce of adulterated products.

FDA is also the Federal lead agency for the development of pesticide analytical methods for food

products. Most of FDA’s research focuses on the

development and modification of multiresidue

methods. FDA developed the surveillance index beginning in 1979 to classify pesticides according to

potential health hazards based on toxicity, prevalence of use, and persistence in the environment.

The index was designed to plan monitoring programs and has helped to prioritize research on

methods for pesticides not detected by multiresidue

methods.

USDA also conducts some research on analytical methods, but most of this work focuses on adapting multiresidue methods for special characteristics of meat and poultry samples (11).

Food and Drug Administration

FDA’s pesticide monitoring program has been designed to accomplish FDA’s legislative mandate under the FDCA within available resources to enforce

EPA pesticide tolerances, and enforce the adulteration provisions of the Act (12). The two primary

objectives of the pesticide monitoring program are

1) to enforce pesticide residue tolerances established

by EPA, and to determine the incidence and level

of pesticide residues in the food supply. FDA’s monitoring program has two major components: general commodity monitoring and the total diet study.

Only multiresidue methods are used for routine testing in these programs. Single residue methods are

reserved for work targeted for a specific pesticide.

The general commodity monitoring program

component involves sampling on an “as shipped”

basis, raw agricultural commodities, processed

foods, and animal feeds. The samples are analyzed

for the purpose of enforcing tolerances established

by EPA, and for determining the incidence and

levels of residues (13). Although an express purpose

of the FDA commodity sampling program is to determine the incidence and levels of residues in commodities, the program is incapable of providing data

that can be used to estimate the general rate of

residues that violate pesticide tolerances. This is because there is no statistically valid plan by which

samples are collected. In fact, a sampling plan that

would make such an estimate possible has not been

studied or compared with the plan in use. Some observers of the program have suggested that the sheer

size, mobility, and decentralized nature of the U.S.

food supply would make it impossible to collect a

random sample of the food supply for pesticide analysis, even if the means were known (14).

FDA’s sampling plan is made up of a set of guidelines to help officials in the district offices determine where to direct their inspection resources,

some guidelines provide commodity-specific quotas,

e.g., “collect 12 egg shell samples, ” others are more

general, “based on local usage, collect agricultural

products for malathion analysis” (15). The plans

specify that the sampling plan should remain flexible so that resources can be shifted to meet special

needs that arise (16). Ultimately, the number of samples collected and analyzed for pesticide residues

in a district is determined by available resources

in that district. Pesticide monitoring must compete

for resources in the districts with other significant

public health functions, and sampling plans are

sometimes derailed by emergency situations (e.g.,

a product tampering incident). In addition, even

when resources are available, some guidelines are

difficult to implement because of inadequate data.

For example, the guideline noted above which

directs testing pesticides based on local use patterns, is reliant on “detective work” done at the local level because little data is available on pesticide

use patterns.

The total diet study (TDS) involves collecting a

“market basket” of food samples several times per

year in several geographic regions of the country,

then analyzing the foods in a ready-to-eat form. The

TDS is used to estimate dietary intake of selected

pesticides by various U.S. age-sex groups (17). The

design of the TDS provides a “snapshot” estimate

of public exposure to those pesticides detected by

the analytical methods used in the study. FDA relies on the TDS to make judgments about the public health risk presented by pesticide exposure

through food.

U.S. Department of Agriculture/Food

Safety and Inspection Service (FSIS)

Unlike the pesticide program of the Food and

Drug Administration, the legislative mandate of

USDA is not just one of enforcing pesticide tolerances in food (meat and poultry) or prosecuting

those who engage in commerce of adulterated products. Instead, Congress has prescribed a system of

ante-(18) and postmortem (19) inspection whereby
222

meat and poultry products are affirmatively certified by USDA to be wholesome and in conformance

with residue limits (20), The program is sometimes

described as “continuous inspection.” An explicit

goal of both the meat and poultry antemortem inspection provisions is to prevent the entry of

adulterated meat or poultry into commerce (21).

USDA has implemented its inspection program by

stationing Federal inspectors in meat and poultry

slaughtering and processing facilities. USDA inspectors visually inspect animals and carcasses and

collect tissue samples for analysis of chemical

residues, including pesticide residues.

The USDA pesticide program is part of its National Residue Program, which also targets residues

of animal drugs and environmental contaminants

in meat and poultry. The program has been revamped

in recent years to focus monitoring activities on pesticide residues according to hazard and estimated

exposure (i.e., risk) (22). The program has three components: monitoring, surveillance, and exploratory

projects. The focus of the monitoring program is

to profile information on the occurrence of pesticide residue violations in specified animal populations on an annual national basis and to form the

basis of enforcement actions (23). Samples are

selected on a statistically random basis. Pesticides

selected for analysis are based on an assessment of

risk and the availability of an analytical method that

is suitable for regulatory purposes (24). USDA tests

for only those compounds that can be detected and

quantified, and for which all metabolizes can be

identified by a practical analytical method. USDA

has acknowledged that because of the large number of potential residues that may occur in the food

chain, practical methods are not available for many

compounds of interest. USDA has defined “practical methods” to be those that 1) require no more

the 2 to 4 hours of analytical time per sample, 2)

require no instrumentation not customarily available in laboratory devoted to trace drug or environmental analyses, 3) have a minimum proficiency

level at or below the established residue limit (e.g.,

tolerance), 4) have a quality assurance plan, and 5)

have undergone an interlaboratory validation study.

Like FDA, USDA relies on multiresidue methods.

The surveillance program is designed to investigate and control the movement of potentially adulterated meat and poultry products. Samples are collected in a non-random, selective fashion directed

at carcasses believed to be adulterated because of

information obtained through investigation or

through the monitoring program. The surveillance

program is sometimes activated to follow the product of a particular supplier who was responsible

for violations in the past. The program gives USDA

the ability to trace problems to their source and take

steps to prevent recurrence.

Exploratory projects can be likened to a research

effort designed to examine a particular problem. Exploratory projects are sometimes used to evaluate

new methods of monitoring or to study the occurrence of residues for which no acceptable limit (e.g.,

tolerance) has been established.

The design of the USDA program has enabled the

agency to sample a statistically representative sample of the U.S. meat and poultry supply for pesticide analysis. This contrasts distinctly with FDA’s

commodity monitoring program, in which the actual sampling decisions are made on an ad hoc basis by inspectors in the field within the broad guidance of the pesticide sampling plans and within

resource and information limitations. However, because the regulatory burdens placed on both agencies limit the resources that can be devoted to pesticide analysis, both agencies have opted to rely on

multiresidue methods almost entirely. Therefore,

those pesticide residues not detected by multiresidue methods generally escape without detection by

any method. This contrast illustrates that in two

agencies, with vastly differing legislative mandates,

the capabilities of existing methods have been key

determinants of the scope, limitations, and effectiveness of monitoring programs,

Federal Pesticide Monitoring

Program Evaluations

Because analytical methods significantly influence the very nature of pesticide monitoring programs, improving methods has been viewed as a

critical requisite of the programs in general. In the

late 1970s, the Subcommittee on Oversight and Investigations of the House Committee on Interstate

and Foreign Commerce, as well as the General

Accounting Office, investigated the Federal pesticide monitoring program administered by FDA.

Among the recommendations the subcommittee

made was that FDA develop analytical methods to

detect more pesticides, and to focus on methods that

could be performed more quickly (25).

In FDA’s own landmark study of ways to improve

the pesticide program (26), FDA emphasized the importance of a “strong, continuously well-supported

and closely coordinated analytical methods development program” (27) to the overall effectiveness

of pesticide monitoring. The FDA study group high-
223

lighted the need for practical analytical methods,

so that they could be used to handle the volume of

samples necessary in a regulatory program, The

group also suggested that research efforts focus on

pesticides not detected by available methods, yet

of concern because of toxicity and prevalence of

use in agriculture (28).

The study group emphasized that for pesticides

not detected by multiresidue methods” . . . research

is needed on other kinds of surveillance analytical

methodology to reduce the overall time and complexity of analyses,’’ Among the specific research projects suggested was the study of rapid

bioassay screening tests that would indicate whether

further residue analysis was needed by a more complex GLC method (30).

Although FDA reprogrammed resources to focus

on these objectives, budgetary constraints and

agency commitments to other public health needs

curtailed the reprogramming possible (31).

Several recent studies of the Federal pesticide programs have stimulated interest in pesticide analytical methods. Among the most important of these

was a 1986 study of the General Accounting Office

(GAO) (32), which reported that there was a significant gap between the number of pesticides that

could potentially be found in food and the number

that could be detected practicably with the multiresidue methods being used in the pesticide monitoring program operated by the Food and Drug

Administration (FDA) (33). FDA was found to rely

on five multiresidue methods. The scope of coverage of each method ranges from 24 to 123 different

residues. Together, the tests are capable of detecting 203 different pesticide residues.

GAO reported that the most serious limitation of

the methods was that they could detect only 40.9

percent of the estimated 496 different pesticides that

potentially could be found in food (34). Furthermore, the methods could detect approximately 64

percent of the estimated 316 pesticides for which

EPA has established food tolerances and are either

currently registered for use on food products or persist in the environment and appear in food despite

cancellation or suspension of food uses (35). Although single residue methods maybe used to detect the estimated 59.1 percent of pesticides not detected by the multiresidue methods, as a practical

matter, they are not used because the inefficiency

of the methods cannot be absorbed in the program

given resource constraints. GAO reported that pesticides not detected by the five multiresidue methods are not routinely monitored.

Although multiresidue methods detect a substantial number of pesticides of health concern, among

those not monitored because of the limitations of

existing methods are 33 of 81 of those pesticides

identified in FDA’s Surveillance Index (36) as being of high priority for routine monitoring (37).

Recognizing the relative cost-efficiency of multiresidue over single residue methods, GAO recommended that FDA expand the number of pesticides

that can be detected by multiresidue methods and,

until “comprehensive capability” exists to test for

most pesticides, conduct more testing of pesticides

not detected by multiresidue methods (38).

The manner in which limitations in analytical capability restrict the effectiveness of monitoring was

highlighted in two recent studies of the USDA National Residue Program. In a 1985 study, the National Academy of Sciences (NAS) made broad recommendations that the National Residue Program

be readjusted to direct inspection to reflect assessments of relative chemical risks and to emphasize

residue prevention. NAS also advised, “[t]he analytical methods used must be appropriate to the task.

. . .The testing program will require substantial support for research, including the development of

more accurate, more sensitive, and less expensive

tests as well as tests for new hazards’’.

A 1987 GAO report pointed out that, as in the FDA

program, a gap existed in the USDA program between the possible residues in food and the scope

of practicable testing methods. GAO recommended

that USDA systematically assess the status of methods for detecting harmful chemicals in food to provide a basis for deciding on the additional research

needed to develop more effective methods (40). In

addition, GAO echoed the advice of NAS that greater emphasis be given to new methods development

including rapid, inexpensive screening tests to detect an array of hazardous compounds (41).

The issue of pesticide analytical methods was also

the focus of a 1987 report of the Congressional Research Service (CRS) (42). That report “considered

whether new and relatively inexpensive rapid analytical methods based on such biological reagents

as enzymes (e.g., enzyme bioassays) and antibodies (immunoassay) might have applications supplementary to those of the multiresidue methods

used in the FDA monitoring program. The report

concluded that enzyme bioassays may offer a relatively inexpensive screening method to use in identifying foods free of certain pesticides (negative results). CRS considered the potential applications of

immunoassay to include uses as single residue
224

methods, or chemical class-specific screening methods. Also, immunoassay were believed to hold

promise as small-scale multiresidue methods. Some

of the tests being designed were considered simple

enough to be performed by relatively unskilled persons in the field. However, CRS noted that a policy

decision to incorporate rapid test methods into the

monitoring program, particularly as screening

methods, would have implications for the cost and

design of the monitoring program because screening is not a regular part of the current monitoring

program (43).

The critical position of analytical methods in pesticide monitoring programs has been recognized

repeatedly in evaluations of Federal programs. Each

study that has recognized the limited scope of the

existing, practical multiresidue methods, has recommended that more research dollars be devoted

to expand the scope of practicable methods. Several studies have suggested the need for less expensive and more rapid methods to cover pesticides

not detected by multiresidue methods. NAS and

CRS have suggested that rapid screening tests may

serve a valuable function in pesticide monitoring.

Analytical Technology and Program

Deslgn

In focusing on analytical methods as a technical

issue bearing on the effectiveness of pesticide monitoring primarily in terms of how many pesticides

can be detected, most policy analyses have treated

methods as merely tools used to reach a predefined

objective. This perspective obscures the fact that

analytical technology serves to define program design and goals.

Analytical methods help to define program design and goals in several ways. First, the scope of

pesticide coverage and limits of detection of analytical methods define what is and what is not detected in a pesticide monitoring program. Second,

the complexity of the method influences who is able

to do the testing and what kind of equipment and

facilities are needed. Third, the level of confidence

in the reliability and validity of the test results influences for what purposes they are suitable. Finally, the resources needed to run the test influence

how many tests can be run within fixed resources.

The cost of the method is influenced by a variety

of factors including its complexity and whether it

provides opportunities for economies of scale.

The development of gas chromatography (GC)

during the late 1950s, has had a significant impact

on the design and goals of Federal pesticide programs. GC technology is the foundation upon which

modern GLC and HPLC multiresidue methods were

developed. Before GC was available, analytical

chemists had to use such relatively unsophisticated

pesticide detection methods as calorimetry and paper chromatography, which provided limited quantitative information. The GC technology transformed analytical capabilities because it provided

reliable quantitative measures of chemicals and

pressed the limits of detection continually lower.

GC was first promoted as a useful method of detecting chemical subunits of fats, known as fatty

acids. However, pesticide residue chemists soon

adapted the technology for pesticide analysis, and

eventually developed the broad scope multiresidue

methods currently used in monitoring programs.

The timing of the GC discovery was significant.

The period of the 1950s and early 1960s was a watershed period for both the analysis and regulation

of chemicals in food (44). The 1954 Miller Amendments to the Food Drug and Cosmetic Act (FDCA)

for the first time established in the law the concept

of scientifically determined tolerances as a basis

for restricting the sale of foods containing pesticide

residues (45). The 1958 enactment of the Food Additive Amendments to the FDCA included the

precedential Delaney clause (46), which reflected

the view prevailing among scientists at the time,

that for at least some health risks of chemicals, particularly carcinogenicity, no “safe” level of exposure could be defined. This view accentuated the

role of chemical detection and fostered efforts to

press the capabilities of analytical chemistry to ever

lower limits of detection.

In 1962, as the Miller Amendments were being

implemented and the potential of GC being explored, Rachel Carson’s influential book Silent

Spring, (47) was published. The book highlighted

concerns about the health and environmental consequences of the organic pesticides (organochlorine

and organophosphorus compounds) developed during World War II. The charges the book made about

the accumulation and persistence of such pesticides

as dichloro-diphenyl-trichloro-ethane (DDT) in the

fat component of foods, including milk, gained the

attention of regulators at FDA (48). As regulators

made these organic pesticides a monitoring priority,

they found GC to be particularly well-suited to detect these classes of chemicals.

The characteristics of GC worked to help shape

the design of pesticide monitoring programs. For

example, the methods had to be performed in the

laboratory, by highly skilled residue chemists. These

features meant that food samples collected had to
225

be sent to equipped laboratories, and results were

not available for several days. The complexity of

the system caused the analysis of each sample to

be relatively costly.

Although improved efficiency was gained through

the development of GLC and HPLC multiresidue

methods, these are still laboratory methods that

must be operated by highly skilled staffs. The limited

scope of multiresidue methods also has served to

define goals of pesticide monitoring. Organic pesticides have remained a priority in the monitoring

of food residues. However, because of the health

and environmental dangers presented by the early

organic pesticides, many have been phased out of

use, Newer pesticides are of more diverse classes

and have been designed to degrade more quickly

into breakdown products to avoid environmental

persistence and accumulation. The changes in pesticide formulation have increased the number and

chemical class diversity of compounds to be analyzed in food, significantly increasing the scientific

task of monitoring them. Many of these chemicals

cannot be detected by the practical multiresidue

methods being used in monitoring programs (49).

Residue problems have gradually shifted outside the

direct focus of multiresidue methods. The very

methods that once provided the means by which

regulators could rise to meet the challenge of monitoring pesticides in food now limit their ability to

do SO.

The limitations of the methods have, in turn,

served to help shape the goals of monitoring programs.

As noted above, because of resource constraints that

prevent the use of single residue methods, both FDA

and USDA have focused their monitoring efforts

on those pesticides detected by multiresidue methods. USDA in particular has articulated as a premise of their monitoring programs that a pesticide

will not be selected for monitoring in the National

Residue Program unless a practical analytical

method exists to detect it (50). Multiresidue methods have thus influenced both the design and the

goal of the monitoring program. In this sense, the

analytical technology of multiresidue methods has

ascended beyond the role of a tool to accomplish

a policy objective to one that helps define the policy objective.

Of course, analytical technology is not the only

variable that influences the design of monitoring

programs. The significant differences in the legislative mandates of FDA and USDA are largely responsible for the enforcement focus of FDA’s commodity sampling program as distinguished from the

certification focus of USDA’s monitoring program,

as a component of the meat and poultry inspection

system (although both programs serve enforcement

purposes). In addition, the legislative mandate of

an agency may also influence the technologies it

adopts into its program by defining certain problems as within the jurisdiction of that agency. For

instance, USDA has in recent years incorporated

rapid screening tests for certain animal drugs, e.g.,

the “sulfa-on-site” test into the meat inspection program, These tests are heIpful to USDA in achieving its mandate of not only enforcing drug residue

limits, in which case they must be confirmed with

a more sophisticated analytical method, but also in

preventing the entry into commerce of food containing illegal residues by obtaining test results

quickly. FDA may have a similar interest in on-site

test results if the mandate of that agency were expanded to require FDA to prevent food containing

illegal residues from entering into commerce.

While the studies discussed above (excepting the

CRS report) have focused on a range of problems

afflicting Federal pesticide monitoring programs,

the recommendations regarding methods research

have arisen from concern about the gap in pesticides potentially present in food and the coverage

of multiresidue methods. Some studies have made

vague reference to the costly nature of pesticide

analyses (e.g., GAO Livestock Report; NAS Meat

Inspection Report) and have implied a need for less

expensive and more rapid test methods. However,

the program studies have focused little attention on

the possible relationship between other fundamental problems in pesticide monitoring programs and

the currently used battery of analytical methods.

Some additional problems of Federal pesticide

monitoring programs that have been recently documented include the following list.

1. The public is exposed to foods sampled and

found to contain violative pesticide residues because the food passes into commerce while the samples are shipped to central laboratories, analyzed,

and results reported (51, 52, 53),

2. Time delays and sample backlogs in FDA laboratories expand the time it takes to obtain analytical findings (54).

3. Because of limited program resources, a relatively small portion of the domestic food supply (no

estimate available) and approximately 1 percent of

imported food shipments are analyzed by FDA (55,

56),

Because most studies of Federal pesticide programs have not focused directly on the possible linkage between analytical methods and monitoring

program design, the possible role of analytical meth-
226

ods in addressing the above problems has not been

highlighted.

As the future of pesticide analytical methods development is charted, there is a danger in focusing

only on strategies to fill the gap between potential

residues in food and residues detectable by multiresidue methods. The so-called gap has been defined using current analytical technology as a reference point, rather than program needs generally.

To focus on the gap alone propagates the limitations

of the current program and the technology that has

helped to define it to the arena of research objectives. Such a focus fails to consider the opportunities analytical technology may offer to improve pesticide programs in more fundamental, structural

ways. Rather, it may inform judgments about the

direction of future methods research to consider

what the future of pesticide monitoring programs

should be. The span between that goal and the status of current pesticide programs is a truer estimate

of the “gap” that represents current program needs.

Examining the ability of current analytical technology to meet those needs within realistic estimates

of program resources will help to suggest an appropriate research strategy.

Questions to Consider as a Research

Strategy is Defined

As research priorities are established for pesticide analytical methods development, it maybe useful to consider the following questions.

Studies of Federal pesticide monitoring programs

not only have revealed a gap in detectable pesticides

but also some structural problems that have been

generated by a system that requires food samples

to be sent to centralized laboratories for analysis.

There, even a relatively small number of pesticide

samples can add to and become enmeshed in laboratory backlog and delay. Can analytical methods

be designed so that they can be performed in the

field?

The studies also reveal a system in which many

of even those relatively few foods that are sampled

and found to be violative, are nevertheless consumed because there is no rapid way to identify

foods containing illegal pesticide residues. Can analytical methods be developed to provide on-site

results?

Reports show a system (FDA) in which resources

run out after only spot-check sampling is done. Can

inexpensive (e.g., screening) methods be developed

so that more sampling and analysis can be performed assuming fixed resources? Can more expensive laboratory methods be reserved for confirming results of screening tests?

In addition to considering how methods development might address problems that have been

identified inside existing monitoring programs, it

also may be instructive to consider the assumptions

of the existing Federal program.

Analytical methods have become a limiting factor in the enforcement of laws concerning pesticide

residues in food. The premise of the pesticide registration system is that registrants will supply to regulators the analytical means to enforce a condition

of registration, i.e., acceptable food residues. As discussed above, the current method requirement imposed on registrants has not fulfilled this objective.

This fact spawns several policy questions regarding analytical methods development,

Would a requirement that the method submitted

by a registrant be useful to regulators be workable?

Would a required contribution by registrants to a

Federal research fund for the development of practicable methods satisfy the need for such methods?

What resource commitment to analytical methods development would be necessary to keep pace

with the advent of new pesticides?

Does the premise of the method development requirement for registrants expect more from technology than can feasibly be delivered?

Even if the scope of analytical methods were

broad enough to detect all possible residues, how

meaningful would it be if scaled-up affordable sampling with rapid analytical results were unavailable?

From an ideal public health perspective, what

should be the food sampling goal? What level of resources would be needed to achieve this goal, given

current analytical technology? What would be the

impact on cost if inexpensive screening tests could

be used in the field to detect residues and laboratory methods reserved for confirmation? Can an

ideal level of sampling be achieved assuming the

use of screening tests and fixed Federal resources?

If industry (e.g., the fresh produce industry, the

food processing industry) obtained private certification of the conformity of their products with pesticide requirements through the use of private analytical testing, what impact would it have on the

goals for analytical methods development?

What are the regulatory implications of developing inexpensive, rapid, and simple pesticide analytical methods that may be used by members of

the public?
227

Do the limitations of pesticide monitoring programs and methods suggest a need for enforcement

policies that focus more attention on residue control than on residue detection?

References

1. Whitaker, A. H., “A History of Federal Pesticide

Regulation in the United States to 1947,” p. 49-

50 (dissertation, Emory University, 1979). (Dr.

Haywood was also head of the insecticide and

fungicide section of the Association of Analytical Chemists.)

2. See, e.g., Montgomery, A. America’s PesticidePermeated Food, Nutrition Action Health Letter, v. 14, June 1987. p. 5 (“Not all pesticides

are unsafe. But some can cause cancer, birth

defects, heritable genetic mutations, and nerve

damage. Clearly, we need to spend more money

and devote more resources to improving our

testing procedures for detecting hazardous

residues.”)

3. Mr. Pasquale Lombardo, Division of Contaminants Chemistry, Food and Drug Administration, priviate interview, Wash., D. C., Jan. 26,

1988.

4. Both GLC and HPLC operate on a similar basic

principle. A chemical mixture (food sample extract) is injected into the portal of specialized

chromatographic equipment that contains a

“separating column. ” The separating column

permits pesticides to pass through it at different rates based on differences in their physical

and chemical properties. As each chemical

reaches the end of the column, each is in turn

sensed by a detector, and a printer displays the

chemical as a “peak” on a chromatographic

print-out. Chemicals can be identified by comparing the time interval between sample injection

and the appearance of a peak with the intervals

of known standard chemicals. The amount of

a chemical is determined by measuring the size

of a peak.

5. The pesticide analytical methods in use in monitoring programs are detailed in U.S. Library of

Congress, Congressional Research Service. “Pesticide monitoring Program: Developing New

Methods to Detect Pesticide Residues in Food,”

by Sarah E. Taylor, Apr. 24, 1987.

6. The most costly part of pesticide analysis by

GLC or HPLC, whether a single or mult-residue

method, is in the perparation of the sample for

injection into the chromatography. Consequently,

because multi- and single residues methods require similar sample preparation, and a multiresidue method provides methods information

on many pesticides, multiresidue methods are

considered more resource efficient that single

residue methods. However, the cost of a multiresidue analysis costs up to $200 per sample

of fruit, vegetable or grain, while fatty foods, including milk and fatty fish cost approximately

$320 per sample analysis. Shroff, A. Regulatory

Affairs Division, Food and Drug Administration, personal communication, Apr. 16, 1987.

7.7 U.S.C. Sec. 135a(c)(5)(d). “Unreasonable adverse effects on the environment” is defined as,

“any unreasonable risk to men or the environment, taking into account the economic, social,

and environmental costs and benefits of the use

of any pesticide. ” 7 U.S.C. Sec. 136(bb).

8. EPA has failed to company fully with the above

described requirements of FIFRA. See, for example, General Accounting Office. Pesticides:

EPA’s Formidable Task to Assess and Regulate

Their Risk. GAO/RCED-86-125. Washington,

DC, 1986. p. 92-100. Registration processes are

discusses in U.S. Library of Congress, Congressional Research Service, “Pesticide Regulation:

Legislative Debate About FIFRA in 1986, ” by

Aidala, J. May 11, 1987. p. 5. See also Conner,

J.D. et. al. Pesticide Regulation Handbook.

(Washington, DC, 1987.)

9.40 C,F.F. 158.125.

10. The EPA validation should be distinguished

from the formal validation required by the Association of Official Analytical Chemists (AOAC)

for a method to gain official status (the “gold

standard” for analytical methods). AOAC requires that at least six laboratories validate a

method. The purpose of validation by AOAC criteria is to assure that the method is capable of

performing as intended and that the results of

an analysis are of acceptable accuracy and precision. See Hill, K.R. and P.E. Corneliussen,

“Validation of Official Methods, ” Analytical

Methods for pesticides and Plant Growth Regulators, v. XV, Academic Press, New York, 1986.

However, the AOAC validation method takes

several years and is so resource-intensive, requiring the attention of the limited number of

expert residue chemists in the U. S., that many

EPA officials consider it an unworkable systems

for their regulatory purposes. Dr. Francis Griffith,

Residue Chemistry Branch, Environmental Protection Agency, personal communication, Feb.

23, 1988. (557-0826).

11. Discussed in U.S. Library of Congress, Congres-
228

12.

13.

14.

15!

16.

17.

18.

19.

20.

21,

22.

23.

24.

25.

sional Research Service. “Pesticide Monitoring

Program, Developing New Methods to Detect

Pesticide Residues in Food,” by S.E. Taylor,

Apr. 24, 1987.

See 21 U.S.C. sec. 342 (a)(2)(C), 374(c)-(d).

Reed, D., P. Lombardo, J. Wessel, L.A. Burke

and B. McMahon. Division of Contaminants

Chemistry. The FDA Pesticide Monitoring Program, J. Assoc. Off. Anal. Chem., v. 70, no. 3,

1987. p. 59.

Duggan, R.E. and H.R. Cook, “National Food

and Feed Monitoring Program,” pesticides

Monitoring Journal, 5:37, June 1971.

Food and Drug Administration, Compliance

Program Guidance Manual, Pesticides and Industrial Chemicals in Domestic Foods (FY88/89),

Oct. 1, 1987.

Ibid.

CDP Associates. Assessment of FDA’s Total Diet

Study, Contract HHS-100-82-0076. 1982.

Poultry Products Inspection Act, 21 U.S.C. sec.

455(s); Meat Inspection Act, 21 U.S. C. sec. 603.

Poultry Products Inspection Act, 21 U.S.C. sec.

455(b); Meat Inspection, 21 U.S.C. sec. 604.

See Poultry Products Inspection Act, 21 U.S.C.

sec. 455(b) (The Secretary, whenever processing operations are being conducted, shall cause

to made by inpsectors, post mortem inspections

of the carcass of each bird processes.”) Federal

Meat Inspection Act, 21 U.S.C. sec. 604 (“[A]ll

such animals found to be not adulterated shall

be marked, stamped, tagged or labeled as ‘inspected and passed’. . . .“).

Federal Meat Inspection Act, 21 U.S.C. sec. 603

(“For the purpose of preventing the use in

commerce of meat and meat food products

which are adulterated. . . .“ ) Poultry Products

Inspection Act, 21 U.S.C, sec. 455(a) (“For the

purpose of preventing the entry into or flow or

movement in commerce of, or the burdening of

commerce by, and poultry product which is . . .

adulterated.)

1987 National Residue Plan, p. 3. A.3.

Food Safety and Inspection Service, United

States Department of Agriculture. Compound

Evaluation and Analytical Capability National

Residue Program Plan 1987, Jan. 1987, p. 2.1.

1987 National Residue Plan, p. 5.1.-5.2.

U.S. Congress. House. Committee on Interstate

and Foreign Commerce, with separate views by

the Subcommittee on Oversight and Investigations. Cancer-Causing Chemicals in Food. 95th

Cong. 2d Sess. Washington, U.S. Govt. Print.

Off., 1978, p. 37-38. Committee Print.

26.

27,

28.

29.

30.

31,

32.

U.S. Dept. of Health and Human Services. Food

and Drug Administration. Public Health Service, Study Group on FDA Residue Programs.

FDA Monitoring Programs for Pesticide and Industrial Chemical Residues in Food. June 1979.

p. 57 [1979 FDA Study Group Report.]

1979 FDA Study Group Report, p. 58-59.

1979 Study Group Report, p. 58-59. (FDA developed the surveillance index system as a response

to this identified goal.)

1979 FDA Study Group Report, p. 57.

Ibid.

See, e.g., U.S. Department of Health and Human Services, Food and Drug Administration.

Public Health Service. Report on Proposals to

Improve Control of Pesticide Residues in Imported Foods, Mar. 10, 1982. p. 9.

U.S. General Accounting Office, “Pesticides:

Need to Enhance FDA’s Ability to Protect the

Public From Illegal Residues, ” Report to Congressional Requesters by the Comptroller General of the United States, GAO/RCED-87-7.

Washington, DC, 1986, p. 31-33. [GAO Domestic Food Report].

33. While the GAO report focused on the FDA monitoring program, its conclusions regarding the

adequacy of pesticide analytical methods also

implicates the methods used in the monitoring

program of the USDA because they are similar

in nature to those of FDA’s program.

34. FDA has disputed the relevance of comparing

the scope of coverage of the five multiresidue

methods to the reference point of 496 pesticides.

FDA contends that the number includes pesticides that are not actually in use because registration is pending or has been cancelled, and

it includes pesticides for which all food uses

have been cancelled. They agency has implied

that 230 may be a more appropriate reference

point. Bowen, Otis R., Secretary of Health and

Human Service. Letter to Charles Bowsher,

Comptroller General of the United States, Mar.

20, 1987.

35. Domestic Food Report, p, 31-34.

36, The surveillance index is a system developed

by FDA in 1979 to classify pesticides according

to potential health hazard, The classification

scheme was developed to help the agency put

in priority its monitoring of residues not detected by multiresidue methods and to direct research efforts to develop new methods. Pesticide chemicals undergoing special review at

EPA because of special health or environmental

concerns have been given priority in being clas-
229

37,

38.

39.

40.

41.

42.

43.

44.

45.

46.

sified in the surveillance index. The index lists

pesticides in Classes I, II, and III. with Class

I being of highests priority based upon the toxicity, use pattern (poundage and crop), potential for food residues and persistence in the environment. See U.S. Dept. of Health and Human

Services. Food and Drug Administration. FDA

Monitoring programs for Pesticide and Industrial Chemical Residues in Food; Study Group

on FDA Residue Programs, June 1979, p. 57.

GAO Domestic Food Report, p. 39.

Ibid., p.43.

Food and Nutrition Board, National Research

Council, National Academy of Sciences. Meat

and Pountry Inspection: The Scientific Basis of

the Nation’s Program, 1985. p. 55-56. In USDA’s

report responding to the recommendations of

NAS, it largely attributed its limited methods

to lack of adequate research funding. USDA suggested that it should be taking advantage of its

potential as a significant purchaser of rapid test

methods to stimulate developmental work in the

private sector. U.S. Dept. of Agriculture, Food

Safety and Inspection Service. FSIS Future

Agenda: Response to the NAS Recommendations. June 1986, ch. 11.

General Accounting Office. Imported Meat and

Livestock; Chemical Residue Detection and the

Issue of Labeling, GAO/RCED-87-142 Wash. D. C.,

Sept. 1987. p. 49-50. [GAO Livestock Report]

GAO Livestock Report, p. 38-39.

U.S. Library of Congress, Congressional Research Service. Pesticide Monitoring Program:

Developing New Methods to Detect Pesticide

Residues in Food, by Sarah E. Taylor, Apr. 24,

1987.

U.S. Library of Congress, Congressional Research Service. Pesticide Monitoring Program:

Developing New Methods to Detect Pesticide

Residues in Food, by Sarah E. Taylor, Apr. 24,

1987. p. 33-34.

U.S. Library of Congress, Congressional Research Service. Food Safety Policy: Selected Scientific and Regulatory Issues. IB83158 by S. E.

Taylor and D.V. Porter (regularly updated) p.

2-5.

21 U.S.C. Sec. 346a (amended by Act of July 22,

1954 ch. 559, 68 Stat. 511).

The so-called “Delaney,” or “anti-cancer” clause

was incorporated into the FDCA through the

Food Additives Amendments of 1958, the Color

Additive Amendments of 1960, and the Animal

Drug Amendments of 1968, The essence of each

clause provides that a substance shall not be

deemed safe and shall not be deemed safe and

shall not be approved if it is found to induce

cancer when ingested by man or animal, or if

it is found after tests that are appropriate for

the evaluation of the safety of the substance to

induce cancer in man or animal. The standard

of safety embodied in the anti-cancer clause is

sometimes termed as “zero-risk” or “zerotolerance” standard. See, U.S. Library of Congress, Congressional Research Service. Food

and Color Additives: “De Minimis ” and

“Delaney,” by Sarah E. Taylor. Aug. 5, 1987.

The Delaney Clause applies to pesticide residues

in foods when residues concentrate during food

processing to levels exceeding the tolerance approved on the food commodity. The implications of the Delaney Clause to perticide chemicals was considered in Board on Agriculture,

National Research Council. Regulating Pesticides in Food: The Delaney Paradox. Wash.,

D. C., 1987.

47, Carson, R. Silent Spring. (Boston: Houghton

48.

49.

50

51

52.

53,

54.

55,

56.

Mifflin Co., 1962) (The book was first published

as a series of articles appearing in The New

Yorker.)

Carson, pp. 140, 169, 178.

Discussed in U.S. Library of Congress, Congressional Research Service. Pesticide Monitoring

Program: Developing New Methods to Detect

Pesticide Residues in Food, by Sarah Taylor,

Apr. 24, 1987. p. 16.

1987 National Residue Plan, p. 5.1-5.2.

GAO Domestic Food Report, p. 4.

General Accounting Office. Better Sampling and

Enforcement Needed on Imported Food. GAO/

RCED-86-219, Washington, DC, Sept. 1986. p.

4. [GAO Imported Food Report]

General Accounting Office. Problems in Preventing the Marketing of Raw Meat and Poultry Containing Potentially Harmful Residues.

HRD-79-10, Washington, DC, Apr. 1979,

General Accounting Office. Laboratory Analyses of Product Samples Needs to be More Timely.

GAO/HRD-86-102, Washington, DC, Sept. 1986.

GAO Domestic Food Report, p. 3,

Ibid.
Appendix C

GIossary of Terms

Glossary of Terms

absorption spectrum: a plot of the amount of light

absorbed by a gas, liquid, or solid at particular

wavelengths versus the wavelengths examined.

acetone: a solvent used to extract pesticides from

foods.

acetonitrile: a solvent used to extract pesticides

from foods.

adsorbent: a material that gathers a gas, liquid, or

dissolved substance on a surface in a condensed

layer.

adsorption chromatography: chromatography based

on the interaction between a chemical dissolved

in a solvent and an adsorptive surface, such as

the surface of a diatomaceous earth particle.

affinity: The strength of the interaction between

chemical and antibody. The higher the affinity

of the antibody for the target chemical, the greater

the sensitivity of the immunoassay.

alkali flame ionization detector (AFID): a detector

that measures the presence of nitrogen and phosphorus within a molecule.

alumina: the natural or synthetic oxide of aluminum.

analytical columns: columns that are used to separate chemicals at the microgram level or below.

aromatics: chemical compounds containing one or

more benzene rings.

atomic emission spectrometric detector: a detector

that measures light emitted from atoms, ions or

molecules following excitation by electrical energy,

flame, or high temperatures; see flame photometric detector.

capillary column: long, open tubes ranging from

0.01 to 0.03 inch in internal diameter and from

30 to 500 feet in length; the inside wall of the tube

is coated with a thin film of involatile liquid.

chemiluminescence detector: a detector that measures the emission of light produced by a chemical reaction.

cholinesterase: an enzyme that hydrolyzes choline

esters.

chromatogram: the record obtained from a chromatographic analysis.

chromatography: the separation of mixtures into

their constituents by preferential adsorption to,

and elution from, a solid support.

compliance samples: commodities collected by FDA

when a violation of pesticide residue tolerance

levels is suspected or known.

derivatization, chemical: modification of a chemical, usually by the addition to or modification of,

a functional group to enhance or permit detection of the compound.

detector, chromatographic: a device for measuring

the amount of a chemical following chromatographic separation.

diatomaceous earth: a fine siliceous earth composed

chiefly of the cell walls of diatoms (any of numerous microscopic, unicellular, marine, or

freshwater algae having siliceous cell walls) used

for adsorption chromatography.

distillation: the volatilization or evaporation and

subsequent condensation of a liquid, as when

water is boiled in a retort and the steam is condensed in a cool receiver.

electroactive functional groups: chemical groups of

a molecule that can be oxidized or reduced electrochemically.

electrochemical detector: a detector that measures

the flow of electrons that occurs with the oxidation or reduction of the chemical analyzed.

electron capture detector (ECD): a detector that measures amount and electron affinity of the chemical analyzed.

elute: to remove an absorbed material from an

adsorbent by means of a solvent.

emulsion: an intimate mixture of liquids, one of

which (the disperse phase) is distributed in large

or small globules throughout the other (the continuous phase). The emulsifying agent, the third

component, is present at the interface between

the two liquids,

exploratory projects: a survey done by FSIS to determine if a pesticide not currently detected or

a method not currently used should be included

in a monitoring program.

fixed wavelength UV absorbance detector: a detector that can measure the absorbance of light by

a chemical at one single wavelength.

flame photometric detector: a type of atomic emission spectrometric detector employing a flame

as a source of excitation of the chemical.

Florisil: a diatomaceous earth adsorbent.

fluorometer: an instrument used to measure the intensity of fluorescence produced by a fluorophor,

fluorophor: a molecule or portion of a molecule that

is capable of excitation by high-energy radiation

and will subsequently emit low-energy radiation.

230
231

fouling (detector): contamination of a detector with

material that decreases the sensitivity or stability of the detector response.

gas chromatography: chromatography in which the

substance to be analyzed is vaporized and diffused along with a carrier gas through a liquid

or solid adsorbent for differential adsorption.

gel chromatography: the separation of molecules on

a column on the basis of size following their

movement into and out of, or their total exclusion from, pores in the gel column.

Hall microelectrolytic conductivity detector (HECD):

a detector that measures the presence of halogens (e.g., Cl, Br), sulfur or nitrogen in a molecule.

hapten: a chemical compound so small that it must

be conjugated to a larger molecule before it can

stimulate antibody production.

herbicide: a chemical for killing plants, especially

weeds.

Hill reaction: the evolution of oxygen from a chloroplast in the presence of ferric ion following the

introduction of light.

hybridoma: a cell type produced by the fusion of

spleen cells and myeloma tumor cells which can

produce monoclinal antibodies.

hydrocarbons: any of a class of compounds containing only carbon and hydrogen.

immiscible: incapable of being mixed.

immunoassay: the use of antibodies to identify and

possibly quantify a substance.

integrators: computers used with gas and liquid

chromatography in part to determine a chemical’s retention time and quantity.

ion trap detector (ITD): a miniaturized mass spectrometer used in the detection of gas chromatography eluants.

ionic: pertaining to electrically charged atoms or

groups or atoms.

liquid phase: the chemical that is bound to the inert supporting phase in a gas chromatographic

column that separates the various components

of a mixture placed on the column.

market basket: a selection of foods that represents

the typical diet of a U.S. household.

mass selective detector (MSD): a miniaturized mass

spectrometer used in the detection of gas chromatography eluants.

mass spectrometry (MS): an analytical technique in

which a chemical is broken into fragments with

positive or negative charge(s) and the mass and

relative abundance of these fragments are analyzed to produce a mass spectrum.

matrix: the material in which the chemical to be

analyzed is found, e.g., pesticides in food.

metabolize: a compound produced from another

(known as a parent compound) as a result of physical and chemical processes acting on the original compound.

microgram: 10-6 grams.

mobile phase: the solvent that flows through a chromatographic column.

monoclinal antibodies: antibodies produced by a

single strain of cloned cells (e.g., hybridomas) in

culture.

multiresidue method (MRM): analytical method that

can detect more than one pesticide during an

analysis of a sample.

nanogram: 10-9 grams.

nanometers: 10-9 meters; used to describe wavelengths of light that are used to excite molecules

and that measure the light absorbed by or emitted from an excited molecule,

neutral: having no electrical charge, positive or

negative.

nitrogenous pesticides: pesticides containing one or

more nitrogen atom(s),

oxidize: to combine with oxygen; to take away

hydrogen,

p-value: a measure of the partition characteristics

of a pesticide between two immiscible organic

phases (solvents).

packed columns: hollow tubing ranging in internal

diameter from 2-4 mm and in length from a few

inches to 50 feet, filled with particles coated with

an inviolatile liquid.

partitioning: the process of distributing between

two immiscible solvents so that the pesticide will

appear in one phase and potential interferences

in another, which can then be discarded.

pesticides: toxic chemicals used against insects (insecticides), fungi (fungicides), weeds (herbicides),

and other pests.

Pestrak: FDA computerized data base used to track

whether pesticides can be analyzed using one of

FDA’s five routinely used multiresidue methods.

photo-diode array detector: a detector that contains

several diodes arranged in series that respond

to the characteristic emitted light of the chemical being analyzed.

photo-ionizable functional groups: chemical groups

such as halides (e.g., Cl, Br) that can be removed

from a larger molecule by the effect of light.

photoconductivity detector: a detector that measures

the change in the conductivity of a chemical in

solution produced by the decomposition of the

chemical by light,

photolyze: the degradation or structural transformation of a chemical by light.
232

photoreaction: a chemical reaction caused by the

reaction of the chemical with light.

polar: that chemical characteristic that favors a

chemical’s volubility in water.

polyclonal antibodies: heterogeneous antibodies derived from different B lymphocyte cells in the

serum of a vertebrate.

ppm: micrograms of chemical per gram of material

in which the chemical is found.

preparative chromatography: the use of columns

having the capability of separating milligram or

larger quantities of chemicals.

pyrolysis: decomposition of chemicals under the influence of high temperatures.

qualitative: of or pertaining to the quality or identity of a substance.

qualitative test: identifies a pesticide residue if it

occurs at concentrations above a pre-established

level.

quantitative: of or pertaining to the measuring of

the quantity of a substance.

relative retention time: the time that a compound

is eluted from a chromatographic column expressed relative to that of a standard compound.

resolution: the true separation of two consecutive

chromatographic peaks.

retention data: retention volume (volume of a carrier gas required to elute a compound from a column) and retention time (time required to elute

a compound from a column.

semiquantitative: intermediate between quantitative and qualitative.

semiquantitative test: identifies a pesticide residue

over a pre-established concentration and determines the range of their concentrations.

silica gel: a highly adsorbent gelatinous form of

silica.

single residue method: analytical method that detects only one pesticide during an analysis of a

sample.

size-exclusion chromatography: see gel chromatography.

solid phase extraction: a technique for concentrating chemicals by absorption and subsequent elution from a liquid phase that is chemically bonded

to silica.

solvating power: the ability of a solvent to interact

with a solute to form a solution.

solvent: a substance that dissolves another to form

a solution.

standard: a chemical, of known concentration and

purity, used as a reference substance in analytical work.

Subdivision “O” Guidelines: guidelines provided by

the U.S. Environmental Protection Agency which

describe the data to be submitted as part of the

tolerance-setting process.

supercritical fluid chromatography (SFC): chromatography in which supercritical fluids are used

as the mobile phase.

supercritical fluids (SF): fluids that are more dense

than gases but not as dense as liquids.

support: the material to which the stationary phase

is attached in a chromatographic column.

surveillance samples: for FDA, these are samples

of food and feed that have been collected for general monitoring purposes; for FSIS these are meat

samples suspected or known to violate pesticide

tolerances.

thermionic detectors (NPD and AFID): detectors that

measure those elements in a chemical compound

that are ionized by heated rubidium (NPD; nitrogen and phosphorus are selectively detected)

or by a heated alkali metal (AFID; nitrogen and

phosphorus are selectively detected).

tolerance: the maximum legal level of a specific pesticide residue on a specific type of commodity,

established by EPA.

Total Diet Study: FDA study that monitors the dietary intake of pesticides in a “market basket” of

foods by various age-sex groups in the U.S.

UV absorbance detector: a detector that measures

the absorbance of light in the ultraviolet range

by chemicals moving through it.

unidentified analytical response (UAR): responses

that appear on a chromatogram which do not

coincide with standards of the pesticides or pesticide metabolizes under investigation.

validating: the process by which one chemist or

more test(s) the suitability of a particular method

for collecting analytical data.

validation: the verification that a technology or

method provides useful analytical data and operates within acceptable performance parameters.

variable wavelength detector: a detector in which

a wide range of wavelengths of light can be detected.

violation rate: percentage of samples analyzed that

violate tolerances.

volatile: evaporating rapidly.

wide bore columns: a type of capillary column used

in gas chromatography that has an internal diameter ranging from 0.53 to 0.75 mm.