Laboratory Methods for 

Efficacy Testing of Bed Bug 

Pesticide Products

Kevin J. Sweeney

Senior Entomologist

U.S. EPA Office of Pesticide Programs 

Registration Division, Washington, DC

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

 Crystal City , VA 1
FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

Crystal City , VA

Draft Product 

Performance

Test Guidelines

OCSPP 810.3900:

Laboratory Testing 

Methods for Bed Bug 

Pesticide Products 

2
Outline of the Presentation

• Introduction - Describe the draft 

guideline’s objectives and organization

• Highlight challenges associated with 

guideline development, testing and data 

analysis

• Review each testing method in Guideline 

Sections (h) through (o)

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

 Crystal City , VA 3
Guideline Objectives

• To provide standardized approaches to bed bug 

pesticide product testing to ensure the quality 

and validity of the efficacy data for these types of 

products 

• To provide guidance to data developers on 

acceptable test methods

• To provide test methods that should yield data 

capable of satisfying the product performance 

data requirements for registration of these 

products

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 4
Guideline Scope

• Does not apply to repellent products applied to 

human skin

• Laboratory methods only 

• Labeling not addressed

FIFRA Scientific Advisory Panel 

 March 6-7, 2012

 Crystal City , VA 5
Guideline Organization

• Definitions important to the guideline

• Protocol elements and recommendations

• Completed study reporting

• Specific guidance on laboratory testing

• List of references

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

Crystal City , VA 6
Key Definitions

• Key definitions include:

– MORTALITY and KNOCKDOWN

– PESTICIDE RESISTANCE

– RESISTANCE RATIO

– SUSCEPTIBLE BED BUG POPULATION

– RESISTANT BED BUG POPULATION

– DISCRIMINATING DOSE

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 7
Experimental Design and 

Evaluation Challenges

• When is a bed bug rated or scored as 

“dead”? 

– Mortality vs. Knockdown

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA

Dr. Dániel Bajomi

8
Experimental Design and 

Evaluation Challenges

• Laboratory 

Testing

– Bridging efficacy 

testing to effective 

outcomes

• Field Testing

– Lack of control 

treatments

– No two residences are 

alike

– Residents may be 

exposed to pesticides

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 9
Experimental Design and 

Evaluation Challenges

• Test Parameters: 

– Appropriate 

exposure times

– Appropriate test 

length

– Positive control or 

standard pesticide

– Replication

FIFRA Scientific Advisory Panel 

March 6-7, 2012 Crystal City , VA 10
Test Protocol Development

• Outlines and describes recommendations for the 

inclusion of specific information in study 

protocols

• EPA does not require study protocols to be 

submitted for bed bug laboratory studies but 

recommend that data developers do so

FIFRA Scientific Advisory Panel

 March 6-7, 2012 

 Crystal City , VA 11
Completed Study Reporting

• Outline and describe recommendations

• Lists specific elements and information be 

included

• Makes reference to Good Laboratory 

Practices

• Documenting what was done is 

recommended

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 12
(h) General Methods

(i) Susceptibility

(j) Residual Activity

(k) Repellency

(l) Impregnated Materials

(m) Foggers

(n) Direct application

(o) Ovicides

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

Crystal City , VA 13
General Methods (h) 

• Information in this section of the guideline 

applies to testing methods outlined in 

sections (i) through (o) 

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 14
General Methods (h) 

Test Substance

• The test substance should be the product 

formulation proposed for use. It should be: 

– identified and certified

– tested at the lowest application rate

– tested at the lowest dilution

FIFRA Scientific Advisory Panel 

 March 6-7, 2012

 Crystal City , VA 15
Propose testing 

of five strains

General Methods (h) 

Test Organism 

Cimex lectularius • 1 susceptible

• 1 resistant

• 3 field strains 

from urban areas 

in 3 different 

regions of the 

USA

FIFRA Scientific Advisory Panel

 March 6-7, 2012 

 Crystal City , VA

Photo: Mr. Jonathan Peck

16
General Methods (h) 

Bed Bug Life Stages

• Adulticide Testing

Unfed adult bed bugs with a 

sex ratio of 1:1

• Insect Growth

 Regulators 

Use mixed nymphal 

stages

• Ovicides

Eggs only

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA

17

Whitney Cranshaw, Colorado State University, Bugwood.org

Gary Alpert, Harvard University, Bugwood.org

Dr. Robin Todd
General Methods (h) 

Test Conditions

• Temperature 25°C

• Humidity 50-80%

• Photoperiod 16:8

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 18
(h) General Methods

(i) Susceptibility

(j) Residual Activity

(k) Repellency

(l) Impregnated Materials

(m) Foggers

(n) Direct application

(o) Ovicides

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

Crystal City , VA 19
Susceptibility (i) 

Experimental Design and Evaluation Challenges

• Insecticide resistance

• Method to determine 

resistance

• At what level of 

susceptibility is a 

bed bug strain 

deemed to be 

“resistant”?

• Need to identify 

resistant strains for 

all modes of action

FIFRA Scientific Advisory Panel 

March 6-7, 2012 Crystal City , VA 20

Whitney Cranshaw, Colorado State University, Bugwood.org
Susceptibility (i) 

Experimental Design and Evaluation Challenges

• Insecticide resistance

– Need to establish/identify 

a susceptible strain as a 

benchmark

– LD vs. LT values: 

– 50% vs. 90% vs. 100%

FIFRA Scientific Advisory Panel 

March 6-7, 2012 Crystal City , VA 21
Susceptibility (i) 

Experimental Design and Evaluation Challenges

– Establishing 

Discriminating/Diagnostic 

Dose Concentrations?

FIFRA Scientific Advisory Panel 

March 6-7, 2012 Crystal City , VA 22
Susceptibility (i)

Testing Objectives 

1) To estimate the susceptibility and 

magnitude of tolerance or resistance of 

bed bug strains to pesticides used in 

product testing; and 

2) To determine the discriminating dose 

for testing 

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 23
Susceptibility (i) 

The Experimental Unit

• 500 ml beaker with the bottom covered 

with filter paper. Filter paper was selected 

because the insecticide solution will be 

absorbed evenly

• Application of 50-100 ul of solution or a 

volume that will saturate the filter paper 

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 24
Susceptibility (i) 

Test Beaker and Surface

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 25
Susceptibility (i)

Test Solutions

• Active ingredient test concentrations 

(dose)

– Logarithmic scale (base 10)

– exponential increase in concentration -

0.0001, 0.001, 0.01. 0.1. 1.0, 10.0

– Untreated control - diluent only

– Positive control – different positive control for 

each mode of action. Deltamethrin for 

pyrethroids. FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 26
Susceptibility (i)

Test Parameters

• Number dose levels = 5 or more

• Number of replicates = 10

• Number of bed bugs per replicate = 20

• Exposure period = 24 hours

• Record mortality, correct with Abbott’s 

Formula and express as a percentage

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 27
Susceptibility (i)

Data Reporting

• Protocol amendments and deviations

• Knockdown and mortality

• Results from the probit analysis

• LD50 and LD90 values for each strain and 

treatment

• LD100 recommended

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

Crystal City , VA 28
Susceptibility (i)

Resistance Ratio Calculations

• Resistance ratios should be calculated as 

follows: 

– LD50 (field strain)/LD50 (susceptible strain) = 

RR50

– LD90 (field strain)/LD90 (susceptible strain) = 

RR90

FIFRA Scientific Advisory Panel 

 March 6-7, 2012

 Crystal City , VA 29
Susceptibility (i)

Discriminating Dose

• Discriminating dose selection

(?) x (LD50, 90, or 100 )

FIFRA Scientific Advisory Panel

 March 6-7, 2012 

 Crystal City , VA 30
Susceptibility (i)

Alternative Product Rates

• Alternative approach using product rates:

– 1x product label rate

– 0.5x product label rate

– 0.25x product label rate

– 0.125x product label rate

– 0.01x product label rate

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 31
(h) General Methods

(i) Susceptibility

(j) Residual Activity

(k) Repellency

(l) Impregnated Materials

(m) Foggers

(n) Direct application

(o) Ovicides

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

Crystal City , VA 32
Residual Activity (j) 

Experimental Design and Evaluation Challenges

• Selection of 

representative 

surfaces

• Choice vs. no-choice

• Balanced treatment 

design

• Exposure time

• Length of the test

• Data analysis

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA

33
Residual Activity (j)

Materials and Methods 

• Residual activity is measured in days, 

weeks or months

– Treated panels are aged to test residual 

claim length

– Expose bed bugs to treatments daily, weekly 

or monthly based on claim desired 

– Testing continues until residual treatment 

fails

– Bed bug exposures have varied from 5 

minutes to 5 days or more

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 34
Residual Activity (j)

Materials and Methods 

• Test surface panels should measure 6" x 6 "

– unpainted 1⁄4" thick plywood 

– commercial linoleum tile 

– concrete board 

– 100% cotton sheeting

– medium pile 100% nylon carpet

• An equal number of product and untreated 

control. Treatments should be established on 

the same surfaces 

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 35
Residual Activity (j)

Materials and Methods

• 5 replicates for each treatment surface per 

bed bug strain and test interval

• Testing begins at day 0 and is repeated every 

7 days until failure 

• Panels should be used only once. New 

(aged) panels should be used at each testing 

interval 

• Use bed bugs only once. New bed bugs from 

each strain should be exposed at each 

testing interval

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 36
Residual Activity (j) 

Materials and Methods

• Apply the lowest rate proposed against bed 

bugs

• Apply liquid formulations at a distance of 1 foot 

above the surface

• Dry panel for 3 hours 

• For dusts, apply from a distance of six inches

• Record application volume and weight of active 

ingredient

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 37
Residual Activity (j)

Materials and Methods

• Apply concentrates at the dilution containing the 

lowest percentage of active ingredient

• For ready to use (RTU) spray and dust products:

– establish the volume delivered using replicated trials 

– use the mean volume to make surface applications

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 38
Residual Activity (j) 

Examples of RTU Nozzle Types

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 39
Residual Activity (j)

Test Parameters

• For each surface tested: 

– 5 replicates with 10 bed bugs each

– 500 bed bugs for each strain tested

– Record data points for every surface at every 

test interval 

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 40
Residual Activity (j)

Fixed Exposure Time to Treatments

• 24 hours followed by transfer to clean 

containers. 

• Observations at 15 & 30 minutes; and at 

1, 2, 4, 24, 48, 72, and 96 hours

• Observations continue unless all bed 

bugs in the product treatment die or 

untreated control mortality exceeds 10%

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 41
Residual Activity (j)

Continuous Exposure to Treatments

• No transfer to clean containers

• Observations at 15 & 30 minutes; and at 

1, 2, 4, 24, 48, 72, and 96 hours and 

every 24 hours thereafter

• Observations continue unless all bed 

bugs in the product treatment die or 

untreated control mortality exceeds 10%

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 42
Residual Activity (j) 

Reporting Study Results

• Protocol with amendments and 

deviations 

• Control mortality should be corrected for 

with Abbott’s Formula

• The percentage of bed bugs killed and 

knocked down, exclusively, for every 

treatment at every test interval 

• The length of the test period in hours or 

days

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 43
Residual Activity (j)

Fixed Exposure Time Results

• Report the number of dead, knocked 

down and live bed bugs in each 

replicate at 15 and 30 minutes; 1, 2, 4, 

24, 48, 72, and 96 hours

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 44
Residual Activity (j) 

Continuous Exposure Time Results

• Report the number of dead, knocked 

down and live bed bugs in each 

replicate at 15 and 30 minutes; 1, 2, 

4, 24, 48, 72, 96 hours and every 24 

hours daily

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 45
Residual Activity (j) 

Data Analysis - Fixed Exposure Time

– The significance of differences within and 

between surface treatments

– Difference between treated and untreated 

surfaces 

– Between strain differences

– An analysis of variance (ANOVA) may 

appropriate but other analysis may be 

considered and should be justified 

– KT50 and KT90 values

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

Crystal City , VA 46
Residual Activity (j) 

Data Analysis for Continuous 

Exposure Times 

– Data should be analyzed with a probit 

analysis to determine the LT50, LT90 and 

LT100 as well as KT50 and KT90 values for 

each surface 

– Provide Goodness-of–Fit test results

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

Crystal City , VA 47
(h) General Methods

(i) Susceptibility

(j) Residual Activity

(k) Repellency

(l) Impregnated Materials

(m) Foggers

(n) Direct Application

(o) Ovicides

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

Crystal City , VA 48
 Repellency (k) 

Objectives 

• Testing should be conducted to determine:

– whether or not pesticide residues alter bed bug 

behavior, either by repellence from or attraction to 

pesticide treated areas in a series of choice tests

• Group and individual bed bug responses 

should be evaluated 

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 49
Repellency (k) 

Experimental Design and Evaluation Challenges

• Pesticide residues and 

bed bug behavior

• Harborage construction

• Harborage conditioning

• Length of test period due to 

toxic effects of insecticide 

treatment

• Individual vs. group 

responses

• Choice testing

• Bed bugs who stay outside 

of the harborage

• Repellency

FIFRA Scientific Advisory Panel 

March 6-7, 2012 Crystal City , VA 50
 

Repellency (k)

Experimental Unit

• Test unit should be large enough to fit up 

to three harborages for each replicate 

• Condition filter paper by exposing to bed 

bugs for 24 hours

• Harborage construction - filter paper 

folded into a tent shape to provide shelter

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 51
 Repellency (k) 

Treatments

• Diluent treated control filter paper

• Product treated filter paper

• Diluent treated conditioned filter 

paper

• Product treated conditioned filter 

paper 

FIFRA Scientific Advisory Panel

 March 6-7, 2012

 Crystal City , VA 52
Repellency (k)

Experiments

• Three experiments

– Unit 1. Each replicate should have two pieces 

of unconditioned filter paper. One treated with 

the pesticide product and the other treated 

with the product diluent or water (if the 

product is ready to use)

– Unit 2. The set-up is the same as in Unit 1 

except that filter papers should be conditioned 

FIFRA Scientific Advisory Panel 

 March 6-7, 2012

 Crystal City , VA 53
Repellency (k)

Experiments

• Unit 3. This unit should include:

– an product treated unconditioned filter 

paper

– a product treated conditioned filter 

paper and

– a diluent treated conditioned filter paper 

FIFRA Scientific Advisory Panel 

 March 6-7, 2012

 Crystal City , VA 54
Repellency (k)

Individual and Group Testing

• Individual responses

• 20 replicates of one bed bug each. 60 bed 

bugs for each strain tested

• Group responses

• 5 replicates of 10 bed bugs each. 150 bed 

bugs for each strain tested

• Expose bed bugs for 1 to 24 hours 

FIFRA Scientific Advisory Panel

 March 6-7, 2012

 Crystal City , VA 55
Repellency (k) 

Data Reporting

• Report:

– Protocol with amendments and deviations

– Amount of product applied

– Location of bed bugs in the arena at end of 

test

– Bed bug mortality and knockdown

FIFRA Scientific Advisory Panel 

 March 6-7, 2012

 Crystal City , VA 56
Repellency (k)

Data Analysis

An analysis of data from individual and group 

testing should determine whether there is a 

difference:

– between the treated and untreated harborages 

without conditioning

– between the treated and untreated harborages 

with conditioning

– between the treated unconditioned and 

treated conditioned harborages 

FIFRA Scientific Advisory Panel

 March 6-7, 2012 

 Crystal City , VA 57
Repellency (k)

Conclusions

• Did bed bugs prefer one harborage over 

another? 

• Did the treatments have any effect?

• Is there a difference between the 

individual and group responses?

FIFRA Scientific Advisory Panel

 March 6-7, 2012 

 Crystal City , VA 58
(h) General Methods

(i) Susceptibility

(j) Residual Activity

(k) Repellency

(l) Impregnated Materials

(m) Foggers

(n) Direct Application

(o) Ovicides

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

Crystal City , VA 59
FIFRA Scientific Advisory Panel March 6

-7, 

2012 Crystal City , VA 60
FIFRA Scientific Advisory Panel March 6

-7, 

2012 Crystal City , VA 61
FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA

62
Impregnated Materials (l)

Experimental Design and Evaluation Challenges

• Test design

• Mortality

• Impact on behavior

– Blood-feeding 

inhibition

– Contact irritation and 

avoidance of treated 

fabric

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA

63

Clemson University - USDA 

Cooperative Extension Slide Series
Impregnated Materials (l)

Objectives

• To test the efficacy of a pesticide impregnated 

material product formulations against the bed 

bug. Test outputs: 

• Mortality

• Blood feeding inhibition

• Detect repellency or attraction to the treated 

material in the presence of a host 

FIFRA Scientific Advisory Panel 

 March 6-7, 2012

 Crystal City , VA 64
Impregnated Materials (l)

Experimental Unit

• Tunnel or Tube Assay

• The tunnel will be lined with treated and/or 

untreated material

• A bait chamber will be placed in one end 

of the tunnel. The chamber will contain a 

host or an artificial blood meal system

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 65
FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 66
Artificial Feeding System

FIFRA Scientific Advisory Panel 

March 6-7, 2012 Crystal City , VA 67
Impregnated Materials (l)

Treatments

• One choice test and two no-choice tests

– Treatment 1. Choice test. 

– Tunnel chamber should be divided into two 

sections 

– One-half lined with untreated product fabric or 

textile 

– The other half lined with pesticide 

impregnated product material

FIFRA Scientific Advisory Panel 

 March 6-7, 2012

 Crystal City , VA 68
Impregnated Materials (l)

Treatments

• Treatment 2. The entire tunnel should be 

lined with pesticide impregnated product 

material

• Treatment 3. The entire tunnel should be 

lined with untreated product material 

FIFRA Scientific Advisory Panel 

 March 6-7, 2012

 Crystal City , VA 69
Impregnated Materials (l)

Replication and Exposure

• 5 replicates for each treatment with 20 bed 

bugs per replicate 

• Replace the blood source in every test 

replicate

• Expose bed bugs to treatment for 24 hours

FIFRA Scientific Advisory Panel

 March 6-7, 2012

 Crystal City , VA 70
Impregnated Materials (l)

Data Reporting

• Report:

– The number of live and dead bed bugs for 

each replicate and treatment 

– The percentage of bed bugs killed in each 

treatment

– Bed bug locations in each replicate at 24 

hours

– The number of fed and unfed bed bugs in all 

replicates

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 71
Impregnated Materials (l)

Blood Feeding Inhibition (BFI)

• Reduction (inhibition) of bed bug blood feeding 

relative to the control should be based on the 

following formula:

• (BFI) = 100 (Bfu - Bft)

 Bfu 

BFu = proportion of bed bugs blood fed in untreated control

BFt = proportion of bed bugs blood fed in treatment

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 72
Impregnated Materials (l)

Data Analysis

–Evaluate the effects of the product 

treatment on blood feeding inhibition

–Evaluate the effects of the product 

treatment on bed bug mortality

–Whether bed bugs were repelled, 

attracted, or indifferent to the product 

treatment

FIFRA Scientific Advisory Panel 

 March 6-7, 2012

 Crystal City , VA 73
(h) General Methods

(i) Susceptibility

(j) Residual Activity

(k) Repellency

(l) Impregnated Materials

(m) Foggers

(n) Direct Application

(o) Ovicides

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

Crystal City , VA 74
Foggers (m)

Objective

• To determine the efficacy of pesticide 

fogging product formulations against bed 

bugs 

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 75
Foggers (m) 

Experimental Design and Evaluation Challenges

• Chamber size

• Test cage construction

• Cage placement

• Replication

• Exposure time

• Test length

• Number of untreated 

control replicates

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA

76
Foggers (m)

Chamber and Dosing

• Testing should be conducted in a chamber 

with a volume of 216 cubic feet or greater 

• Test doses for aerosols, misters and fogger 

products should be delivered by an 

automatic dispenser calibrated for the 

proper droplet size and application rate

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 77
Foggers (m)

Treatments and Replications

• There should be a product treatment and 

an untreated control treatment for each 

bed bug strain

• 24 replicates per treatment

• 10 bed bugs per replicate

FIFRA Scientific Advisory Panel

 March 6-7, 2012

 Crystal City , VA 78
Example of a Chamber

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 79
Foggers (m)

Test Cages 

• For each replicate the test cage should be 

a PVC pipe 6 long with a diameter of one 

inch. Each end is covered with a 

removable PVC cap 

• Twenty 1/16 holes should be drilled in a 

random array around the circumference of 

pipe

FIFRA Scientific Advisory Panel

 March 6-7, 2012 

 Crystal City , VA 80
Foggers (m)

Test Cage Placement

• Cages placement for each strain tested:

– on all 4 walls at 3 height levels 

– 2 cages for each height level on each wall 

– 6 cages per wall

– 24 cages total

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 81
Foggers (m)

Exposure and Evaluation

• 2 hour exposure to the treatment

• Ventilate chamber 

• Evaluate treatments and controls at 24, 

48, 72, and 96 hours post-treatment

FIFRA Scientific Advisory Panel 

 March 6-7, 2012

 Crystal City , VA 82
Foggers (m)

Data Reporting

– Mortality data at 24, 48, 72 and 96 hours posttreatment 

– Percentage killed and knocked down for every 

replicate at each observation interval 

– Pooled product treatment mortality data for 

each height level and 

– Corrected mean values for treatments and 

controls

– Amount of product applied

FIFRA Scientific Advisory Panel

 March 6-7, 2012

 Crystal City , VA 83
Foggers (m)

Data Analysis

• The analysis should consider treatment, 

cage height, and bed bug strain effects on 

product efficacy 

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 84
(h) General Methods

(i) Susceptibility

(j) Residual Activity

(k) Repellency

(l) Impregnated Materials

(m) Foggers

(n) Direct Application

(o) Ovicides

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

Crystal City , VA 85
Direct Application (n)

Method Overview

Established methods that are widely used

FIFRA Scientific Advisory Panel

 March 6-7, 2012 

 Crystal City , VA 86
(h) General Methods

(i) Susceptibility

(j) Residual Activity

(k) Repellency

(l) Impregnated Materials

(m) Foggers

(n) Direct Application

(o) Ovicides

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

Crystal City , VA 87
Ovicides (o) 

Experimental Design and Evaluation Challenges

• Ovicidal testing is conducted by many 

laboratories

– Oviposition surface has been unpainted wood 

or filter paper (treated and/or untreated)

• Evaluate oviposition repellency and egg kill

– Direct spray testing has been used

– Many current product residual applications will 

kill nymphs immediately after hatch but not 

eggs

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 88
Ovicides (o) 

Experimental Design and Evaluation Challenges

– Selecting and separating 

female bed bugs

– Egg collection, transfer 

and storage

– Determination of egg kill 

vs. lack of egg viability 

– Control “mortality”

– Test length

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 89
Ovicides (o)

Direct Spray 

• Direct spray test system

– Pieces of egg-laden filter paper should be cut 

into piece that should fit into the test arena

– Up to 6 eggs should be allotted to each well 

or dish

– A metered bench top sprayer is preferred as 

the spray device to ensure consistent 

application volume and even distribution of 

spray particles

– Applications should be made at the lowest 

rate 

FIFRA Scientific Advisory Panel 

 March 6-7, 2012

 Crystal City , VA 90
Ovicides (o) 

Direct Spray Replication

• Direct spray test system

• Minimum of 10 replicates for each 

treatment 

• Minimum of 20 eggs per replicate

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 91
Ovicides (o) 

Contact Kill 

• Experimental Unit 1

– Conditioned filter paper should be used to line 

untreated control and product treatment 

dishes

– 1 female bed bug per dish

– Each dish is a replicate

• 25 replicates per treatment 

–The number of eggs laid on the filter 

paper by each female will be variable

FIFRA Scientific Advisory Panel 

 March 6-7, 2012

 Crystal City , VA 92
Ovicides (o)

Contact Kill 

• Experimental Unit 2 - same set-up as Unit 1 

except bed bugs do not lay eggs on treated 

surface.

– Eggs are transferred to each dish and placed 

on top of the paper - or -

– Egg laden untreated papers can be turned 

upside down to bring eggs into contact with 

the treated surface

– Each dish is a replicate and should have 10 

eggs 

– 25 replicates per treatment 

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

 Crystal City , VA 93
Ovicides (o)

Data 

Reporting

- Exposure time 

is 30 days or 

less. 

- Observe eggs 

daily 

- Record unhatched and 

hatched eggs

FIFRA Scientific Advisory Panel 

 March 6-7, 2012

 Crystal City , VA 94

Gary Alpert, Harvard University, Bugwood.org
FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA

95

Mohammed El Damir, Pest Management, Bugwood.org
Ovicides (o)

Data Analysis

• Compare means between product 

treatments and controls to determine if 

they are significantly different at the end of 

the test period

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 96
Questions and Comments?

97

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA

Photo: Jonathan Peck
Charge to Panel

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 98
Charge 1) Laboratory test methods. The draft 

guidelines describe laboratory test methods for 

evaluating the efficacy of a variety of bed bug 

pesticide products. Please discuss:

(a) Whether, given the objectives and the types 

of products being evaluated, the test methods 

are appropriate to evaluate the efficacy of bed 

bug products with regard to kill and knockdown; 

repellency; pesticide resistance; and 

discriminating dose.

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 99
1) (b) Whether there are additional or alternative 

laboratory test methods beyond those discussed 

in the draft guidelines for testing the efficacy of 

bed bug pesticide products.

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 100
Charge 2) Standardized test system elements 

and conditions (Section (h) pp. 10). The draft 

guidelines describe standard elements and 

conditions that are recommended for each type 

of efficacy evaluation. Please discuss:

(a) The appropriateness of suggested 

environmental conditions (such as temperature 

and humidity). 

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 101
2) (b) The proposed test organism. In particular, 

please discuss:

(i) Whether the testing should be done only with 

one species, the common bed bug, Cimex 

lectularius. Whether it is appropriate to use 

unfed adult bed bugs with a sex ratio of 1:1 to 

test adulticide products.

FIFRA Scientific Advisory Panel 

 March 6-7, 2012 

Crystal City , VA 102
2) (b)

(ii) The advantages and disadvantages of 

including a susceptible strain in every test and 

whether the Harlan strain is the preferred 

susceptible strain.

(iii) The advantages and disadvantages of 

including a resistant strain in every test and how 

an investigator should select a resistant strain or 

strains.

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 103
2) (b) 

(iv) The adequacy of the use of field collected 

bed bug strains from urban areas in three 

regions of the U.S. to represent the variability in 

susceptibility of bed bug populations for 

conducting efficacy evaluations. 

(v) The recommended approach for grouping 

and evaluating the data from the tested bed bug 

strains to assess efficacy of bed bug products. 

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 104
2) (c) The advantages and disadvantages of 

including a positive control in every test. 

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 105
Charge 3) Specific guidance for laboratory studies for 

resistant ratio determination, characterization of bed 

bug strain susceptibility, and discriminating dose 

selection (Section (i) pp.11). The draft guidelines 

propose a method to collect the data necessary to 

calculate a resistance ratio. Resistance ratios should be 

calculated for bed bug populations collected from the 

field in three regions in the U.S. The lethal dose values 

to be used in these calculations are to be derived from 

probit analysis. For pyrethroid insecticides, deltamethrin 

is proposed as the laboratory standard. From the 

collected data and resistance ratio values, the procedure 

recommends an approach to select a discriminating 

dose for a product. Please discuss: 

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 106
3) (a) Which insecticides should be chosen as 

standards for other insecticidal modes of action. 

(b) Whether it is useful to compare the 

resistance ratios of bed bug field populations to 

the resistance ratio for a corresponding dose of 

deltamethrin when evaluating pyrethroid 

insecticides. 

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 107
3 (c) Whether a resistance ratio of 100x is adequate to 

screen field strains for resistance against insecticides 

and if not, what other approaches are recommended for 

detecting resistance in bed bug populations.

(d) What type(s) of data analysis and statistical testing 

would be most appropriate for these data sets.

(e) The discriminating dose selection for products, 

including whether the LD90 value of the least susceptible 

field population is the best value to use as the basis for 

discriminating dose selection. 

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 108
Charge 4) Exposure times to product treatments 

[Sections (j) (pp. 13) and (l) (pp.17)]. For residual 

surface and impregnated material testing, the draft 

guideline proposes two possible approaches to exposing 

bed bugs to pesticide product applications in the 

laboratory: 1) single dose with a fixed exposure time of 

24 hours followed by mortality assessments through 96 

hours unless all bed bugs die or control mortality 

exceeds 10%; and 2) single dose with an exposure time 

that is continuous until all bed bugs die or control 

mortality exceeds 10%. The single dose is generally the 

lowest label recommended dose for the product. Please 

discuss:

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 109
4) (a) Whether the exposure times provide 

sufficient data to measure the efficacy of a 

bed bug pesticide or whether other 

exposure times or testing scenarios should 

be used.

(b) Whether percent mortality values or 

lethal time values should be used as 

endpoints to assess the success of 

product applications and the advantages 

and disadvantages of each one.

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 110
Charge 5) Specific guidance for laboratory 

studies for forced exposure (no-choice) 

residual surface treatment tests (Section (j) 

pp.13). The methods described for testing 

residual surface treatments recommend five 

types of surfaces: unpainted plywood; linoleum 

tile; concrete board; cotton sheet; and medium 

pile carpet. Please discuss:

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 111
5) (a) Whether the experimental unit described in 

the draft guidelines will provide sufficient data to 

show how effectively and how quickly a bed bug 

product knocks down and kills bed bugs, when 

applied as a residual treatment to surfaces.

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 112
5) (b) Whether there is a single surface type that 

could be used as a standard or representative 

surface for testing product residual activity in lieu 

of testing multiple surfaces as recommended in 

the draft guidelines.

(c) Whether there are modifications or additional 

tests that could be recommended to improve 

residual surface treatment testing.

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 113
5) (d) What type(s) of data analysis and statistical 

testing would be most appropriate for these data 

sets.

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 114
Charge 6) Specific guidance for laboratory 

studies to determine if bed bugs are repelled 

by, or attracted to, pesticide product 

residues (Section (k) pp. 15). For pesticide 

product treatments to be efficacious, bed bugs 

must contact pesticide residues. Testing 

methods should determine whether or not 

pesticide residues alter bed bug behavior, either 

by repellence from or attraction to pesticide 

treated areas. Please discuss:

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 115
6) (a) Whether the experimental unit described in 

the draft guidelines will provide sufficient data to 

measure the duration and extent to which a 

pesticide product’s residues repel bed bugs. 

(b) Whether conditioning of experimental bed 

bug harborages is necessary.

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 116
6 (c) Whether individual responses and/or group 

responses should be used to determine whether 

bed bugs are repelled by pesticide product 

residues in harborages. 

(d) What type(s) of data analysis and statistical 

testing would be most appropriate for these data 

sets.

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 117
Charge 7) Specific guidance for laboratory 

studies for testing pesticide impregnated 

material products (Section (l) pp. 17). The 

draft guideline proposes no-choice and choice 

tests for treated materials. Both are to be 

conducted in the presence of a non-human host 

or artificial membrane system to provide a 

source of blood. The proposed tests evaluate 

mortality, blood feeding inhibition, and 

preference for treated versus untreated 

surfaces. Please discuss:

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 118
7) (a) Whether the experimental unit is adequate 

for evaluating mortality and blood feeding 

inhibition following exposure to impregnated 

materials. 

(b) Whether the use of an artificial membrane 

system to simulate an animal host and provide 

blood for questing bed bugs is adequate, or 

whether an animal host is necessary.

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 119
7) (c) Whether the assessment period is adequate.

(d) Modifications or additional tests that would 

improve pesticide impregnated product testing. 

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 120
7) (e) Adequacy of the experimental unit to provide an 

experimental design and adequate data to evaluate 

repellency.

(f) What type(s) of data analysis and statistical testing 

would be most appropriate for these data sets.

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 121
Charge 8) Specific guidance for laboratory 

studies of indoor fogger products (Section 

(m) pp.19). The methods described for indoor 

fogger testing recommend use of an 

experimental unit that includes a 216 cubic feet 

or larger Peet-Grady chamber. The test 

container for bed bugs has 20 holes (1/16" 

diameter) in it to simulate a crack and crevice 

treatment and a bed bug refuge. Please discuss: 

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 122
8) (a) Whether the experimental unit is adequate 

for testing indoor foggers and misters.

(b) Modifications or additional tests that could be 

recommended to improve indoor fogger testing. 

(c) What type(s) of data analysis and statistical 

testing would be most appropriate for these data 

sets.

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 123
Charge 9) Specific guidance for laboratory 

studies for testing ovicidal products (Section 

(o) pp. 22). Efficacious ovicidal products are 

likely to improve the effectiveness of bed bug 

management programs. The draft guidelines 

describe methods for evaluating product efficacy 

against eggs from direct application and contact 

with residual surface applications. Please 

discuss: 

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 124
9) (a) Adequacy of the experimental unit for testing 

ovicidal products. 

(b) Modifications or additional tests that could be 

recommended to improve ovicidal product 

testing. 

(c) What type(s) of data analysis and statistical 

testing would be most appropriate for these data 

sets.

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 125
Charge 10) Please provide comments on the 

overall clarity, accuracy and completeness of 

the draft guidelines: “Laboratory Testing 

Methods for Bed Bug Pesticide Products”.

Please provide any additional comments that 

highlight any areas of the draft guidelines that 

may need to be clarified and note any relevant 

topics that may be missing. Please include 

references to any published literature that could 

help improve the completeness and clarity of the 

draft guidelines. 

FIFRA Scientific Advisory Panel 

March 6-7, 2012 

Crystal City , VA 126