Product Performance Test Guidelines: OCSPP 810.3100: Treatments for Imported Fire Ants


&EPA
United States	Office of Chemical Safety and	EPA 712-C-21-001
Environmental Protection	Pollution Prevention (7101)	January 5,2021
Agency
Product Performance
Test Guidelines
OCSPP 810.3100:
Treatments for Imported
F ire Ants

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NOTICE
This guideline is one of a series of test guidelines established by the Office of Chemical Safety and
Pollution Prevention (OCSPP) [formerly the Office of Prevention, Pesticides and Toxic Substances
(OPPTS) prior to April 22, 2010], United States Environmental Protection Agency (US EPA) for
use in testing pesticides and chemical substances to develop data for submission to the agency
underthe Toxic Substances Control Act (TSCA) (15 U.S.C. 2601, etseq.), the Federal Insecticide,
Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and section 408 of the Federal
Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a), referred to hereinafter as the harmonized
test guidelines.
The OCSPP test guidelines serve as a compendium of accepted scientific methodologies for
research intended to provide data to inform regulatory decisions under TSCA, FIFRA, and/or
FFDCA. This document provides guidance for conducting appropriate tests, and is also used by
EPA, the public, and the companies that are required to submit data under FIFRA. These guidelines
are not binding on either EPA or any outside parties, and the EPA may depart from them where
circumstances warrant and without prior notice. The methods described in these guidelines are
strongly recommended for generating the data that are the subject of the guidelines, but EPA
recognizes that departures may sometimes be appropriate. You may propose alternatives to the
methods described in these guidelines, with supporting rationale. The agency will assess them for
appropriateness on a case-by-case basis.
For additional information about the harmonized test guidelines and to access the guidelines
electronically, please go to https://www.epa.sov/test-suidelines-pesticides-and-toxic-substances.
You may also access the guidelines in http://www.regulations. gov grouped by Series under Docket
ID #s: EPA-HQ-OPPT-2009-0150 through EPA-HQ-OPPT-2009-0159, EPA-HQ-OPPT-2009-0576,
and EPA-HQ-OPP-2011-1017. EPA-HQ-QPP-2017-0693 is the docket number for this guideline.
The contents of this document do not have the force and effect of law and are not meant to bind the
public in any way. This document is intended only to provide clarity to the public regarding existing
requirements under the law or agency policies.
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OCSPP 810.3100: Treatments for Imported Fire Ants
(a)	Introduction.
a.	Scope. This guideline provides recommendations for the design and execution of laboratory and
field studies, the results of which, when combined, are used to evaluate the performance of
pesticide products for the treatment of ants in the Solenopsis saevissima complex, which includes
in part, for the purposes of the guideline, only S. invicta, S. richteri, and their hybrids in
connection with registration of pesticide products under the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.). This guidance applies to products in any
formulation, such as a liquid, aerosol, granular or bait, if intended to be applied for control of
imported fire ant colonies of the above listed species. This guideline does not apply to those
products exempt from FIFRA Registration under 40 CFR 152.25 or to product performance
testing described in other agency guidelines. For example, tests for additional formulations of
products targeting fire ant individuals (rather than colonies), such as direct spray testing and
indoor/outdoor residual applications targeting foragers, can be found in the Product Performance
Test Guidelines OCSPP 810.3500: Premises Guideline for appropriate testing. This guideline
revises OCSPP Test Guideline 810.3100: Soil Treatments for Imported Fire Ants (March 1998).
b.	Purpose. This guideline provides laboratory and field study methods, the results of which, when
combined, are used to evaluate product performance of pesticides against imported fire ant
colonies of S. invicta, S. richteri, and their hybrids and includes statistical analysis and reporting
recommendations.
(b)	Organization of the OCSPP 810.3100 Guideline.
a.	(a) Introduction;
(b)	Organization of the of the OCSPP 810.3100 Guideline;
(c)	Definitions;
(d)	Development of protocols for efficacy studies;
(e)	Review of protocols for efficacy studies;
(f)	Execution of efficacy studies;
(g)	Reporting of completed efficacy studies to the agency;
(h)	Retention of records;
(i)	Specific guidance for field studies for testing area-applied pesticide products;
(j) Specific guidance for field studies for testing mound-applied pesticide products;
(k) Specific guidance for laboratory studies for testing bait products;
(1) Specific guidance for laboratory studies for testing products other than baits;
(m) Specific guidance for laboratory studies for testing Insect Growth Regulator (IGR) products;
(n) References.
b.	General Considerations. Any protocol and/or study developed using this guidance must meet
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the provisions set forth in several statutes and regulations, including, but not limited to, the
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA, 7 U.S.C. 136, et seq.) under which
EPA regulates pesticides. This guideline does not supersede or overrule the regulations governing
research conducted with human subjects such as those contained in 40 CFR Part 26, or any other
Agency regulations. To the extent there are any unintended conflicts between this guideline and
any EPA regulation, the regulation at issue governs.
i. Good Laboratory Practice Standards. Good Laboratory Practice (GLP) Standards set
forth in 40 CFR § 160 apply to laboratory studies evaluating pesticide product
performance. Part 158 specifies that "applicants must adhere to the good laboratory
practice (GLP) standards described in 40 CFR § 160 when conducting studies" [40 CFR §
158.70(b)], However, studies that do not comply with GLP standards may nonetheless be
considered if, in the Agency's judgment, the design and conduct of the study provide
results that are scientifically reliable. 40 CFR §160.12(b) states that with any submitted
research data "[a] statement describing in detail all differences between the practices used
in the study and those required by this part" must be submitted to aid in making that
determination.
ii. State requirements. Investigators and Sponsors should ensure research is conducted in
compliance with any applicable state laws or regulations, which are independent of and
additional to those cited in this guideline.
(c) Definitions. The following definitions are of special importance in understanding this guideline. They
apply only in the context of this guideline and are not intended to be more generally applicable.
a. Active mound; see Mound.
b. Application rate refers to the amount of product applied per unit area or volume (e.g., oz/ft2).
c. Brood refers to the immature members of a colony collectively, including eggs, larvae, and
pupae (Torre-Bueno 1989).
d. Colony refers to a group of related individuals, which constructs nests and rears offspring in a
cooperative manner (Torre-Bueno 1989); mature colonies contain brood.
e. Control refers to when a product kills a fire ant colony for a specified duration of time in field
studies (e.g., > 30 days) and is supported by evidentiary data from laboratory tests for product
performance specifically against the workers, queen and in some cases, brood.
f. Field study refers to a scientific investigation that occurs in the listed fire ant's typical habitat or
dwelling space.
g. Food lure refers to a food item, such as hot dog pieces, used to attract foraging ants.
h. Forager refers to, in social insects, a member of the worker caste that gathers food and brings it
back to the colony (Torre-Bueno 1989).
i. Method of application refers to the way a pesticide can be delivered (applied) to a pest or site.
Examples of application methods include dusts, watered-in granular products, and bait.
j. Monogyne refers to colonies containing a single fertile queen.
k. Mortality refers to ant death. A dead ant is an ant that does not move, even when poked or
probed.
1. Mound refers to the above-ground and below-ground structure constructed for thermoregulation
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of a colony; an active mound contains all or part of a colony. A mound is considered active when
at least 20 workers exit the mound immediately following disturbance (Oi & Oi 2006).
m. Negative control refers to the group of specimens in an experiment that receive no treatment or a
treatment with the diluent only or are given a placebo bait; no response is expected.
Photoperiod refers to the relative amount of time during the day in which it is light or dark
(Triplehorn et al. 2005)
Polygyria refers to colonies containing multiple fertile queens.
Product performance testing refers to scientific studies that are designed to test the effectiveness
of a pesticide product against the specific target pest.
Queen refers to a mated, egg-laying female member of the reproductive caste.
Social form refers to the number of egg laying queens in a colony; a monogyne colony has a
single fertile queen and a polygyne colony has multiple fertile queens. Polygyne infestations may
establish twice the density of monogyne infestations (Porter 1992).
(d) Development of protocols for efficacy studies. Testing pesticides for efficacy against listed fire ants
begins with development of a study protocol that includes both laboratory and field components. General
considerations in developing a study protocol for efficacy studies include scientific design of the study,
data collection, data analysis, and reporting. The product should be tested in a manner that reflects
intended use and that will provide data to support product performance. Both field and laboratory tests
should be used to adequately address efficacy. Laboratory tests provide evidentiary data for product
performance specifically against the workers, queen and in some cases, brood. Each of these topics is
discussed in more detail in the sub-sections below. Additional study-specific considerations can be found
in Sections (i) through (m).
a. Scientific design of study. The experimental methods should be likely to provide a definitive
answer to the research question and should include a detailed description of the experimental
design, addressing topics i. through viii., given directly below.
i. Objectives. For products that kill listed fire ant colonies, the objective of product
performance testing is to determine efficacy at the lowest proposed label rate that
kills/controls a colony. Typically, if a product kills a colony, this includes controls claims.
In all cases, the scientific objective and intended label claims should be stated clearly and
all treated colonies/mounds/nests should be compared to negative control
colonies/mounds/nests that have received no treatment, placebo bait treatment or a diluent-
only treatment.
ii. Test materials and treatments. End-use formulations should be tested using the lowest
labeled application rates for use on listed fire ant colonies. Test materials should be stored
at ambient temperature and humidity for at least one day before use.
1. Products that are area-applied (i.e., applied to an area vs. individual mounds) should
be tested using assessments of foragers when field testing and workers, queen and
brood for the laboratory portion of the testing. Refer to section (i) vi. 1-3 for
specific guidance for area-applied field study endpoints. Refer to section (k)
through (m) for specific guidance for laboratory studies.
2. Products that are mound-applied should be tested using assessments of mounds
when field testing and workers, queen and brood for the laboratory portion of the
testing. Refer to section (j) vi. 1-2 for specific guidance for mound-applied field
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study endpoints. Refer to section (k) through (m) for specific guidance for
laboratory studies.
3. Products that target ant development should be tested using brood, queen, and
workers.
Application rate determination. The test rate in product performance studies is the lowest
application rate on the target pest from a proposed product label, typically expressed as
amount of product per unit area for surface area treatments (e.g., 1 lb/500 ft2). Rates may
also be expressed as amount of product applied per mound (including a maximum amount
per acre, if applicable). The amount of active ingredient applied per unit area should also be
reported. Label rate should match the rate tested in the efficacy study. The application
device used in testing should be the same as the proposed or registered product (e.g., to
support use of a bait station, the bait product should be tested in a bait station). While rates
should be reported in units according to the U.S. traditional systems of weights and
measures, units may additionally be reported using metric system measurements. The
method to measure test rate can vary among studies; however, the following are common
methods of measurement:
1. Measure the amount of test product (e.g., granular bait or dust) applied per mound.
To calculate the amount applied per unit area of the test plot containing the treated
mounds, multiply the amount applied per mound by the total number of treated
mounds on a plot, divided by the area of the plot.
2. Measure the amount of test product added to a bait station. To calculate the total
amount applied per unit area of the test plot containing the bait stations, multiply
the amount applied per bait station by the total number of bait stations on a plot,
divided by the area of the plot.
3. Measure the amount of test product, such as a liquid or granular product, in its
container before and after application, and divide the difference by the unit area
treated.
Testing conditions. During product performance testing in the laboratory, testing may be
conducted at ambient temperature and relative humidity (RH) in the testing arena as long as
conditions are optimal for foraging behavior, such as 82°-91° F (Banks et al. 1981).
Conditions that are too cool or dry will inhibit normal colony activity. In addition,
temperature and RH within the nest cell should also be maintained at conditions optimal for
brood development, such as > 65 % RH and 82°-91° F (Williams 1989). Also, laboratory
colonies should be provided time to acclimate to the photoperiod, as photoperiod is known
to affect foraging behavior (Lei et al. 2019). Environmental conditions for laboratory
testing, including temperature, humidity, and photoperiod during testing, should be
supported by cited literature. The temperature during the test should be kept as constant as
possible because changes can affect the performance of the product treatments. A food and
water source should be provided for all test organisms throughout the study.
Field studies should be conducted in weather conditions that are realistic for use. Extreme
weather (temperature, wind and/or precipitation) should be avoided. Recommendations for
field study test durations differ based on product application (i.e., mound vs area-applied)
and whether an IGR is included in the product formulation. Field study duration for a
mound applied product, where the colony within the mound presumably receives the full
labeled rate, typically follows a > 30-day duration. Area-applied products typically follow a
> 60-day duration because an application is used to treat all colonies within an area.
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Colonies within the treated area may collect different amounts of the applied product due to
variability in sizes of the colonies and their ability to monopolize foraging territory.
Therefore, the treatment effect may take longer to be apparent and/or may dissipate quicker
(i.e., shorter residual effect) in colonies that did not acquire a lethal dose. Likewise, for
products containing an IGR, a > 60-day duration is recommended to observe an effect
unless a longer duration is needed for specific claims, such as colony mortality or a 6-
month control claim.
v. Choice of endpoints. Study endpoints should be appropriate for the specific objectives of
the proposed research and likely to provide a robust answer to the research question.
Endpoints such as mortality should be evaluated at the lowest labeled application rate for
listed fire ant colonies/mounds/nests. The endpoint selected should be claimed on the
proposed label. The following are examples of commonly used endpoints; see specific
study sections (i) through (m) for additional information or variations.
1. Mortality. Observations of mortality should be reported based on recommendations
provided in the specific study design. The number of dead worker ants and queens
in each replicate should be recorded separately at each time point tested, as
practically possible. A mortality count should include only dead ants.
2. Number of active mounds. Numbers of active mounds on plots should be reported
based on recommendations provided in the specific study design. The number of
active mounds in each replicate should be recorded separately at each time point
tested. To determine if mounds are active, the surface of each mound should be
scratched with a pair of forceps or similar object. Alternatively, a narrow probe,
similar to a stem of a field flag (approximately 0.12"), can be inserted into the
mound from 6"- 8" deep.
3. Forager numbers. Numbers of foragers collected at food lures should be reported
based on recommendations provided in the specific study design. The number of
foragers collected in each replicate should be recorded separately at each time point
tested.
4. Presence/absence of brood. Observations of presence/absence of brood should be
reported for all colonies/mounds/nests based on recommendations provided in the
specific study design.
vi. Test organisms. Testing should be conducted with adult listed fire ants unless the product
is intended to target ant development and/or immature stages. All sources of fire ants
should be listed in the study methods along with species.
vii. Representative sampling.
1. Replication. The protocol should fully describe how sample size and replication
were determined, and a power versus sample size analysis should be performed to
support the proposed sample size. Factors that may affect sample size and
replication are the number of treatments, experimental design, the environment
(e.g., different habitat population densities), and heterogeneity in the sample pest
population (e.g., social form). For example, areas known to contain the polygyne
social form may have twice the density of mounds compared to the monogyne
social form (Porter 1992).
2. Rearing, handling, and maintenance of listed fire ants. When applicable, a
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description of the laboratory colony rearing practices should be included. Collection
details and maintenance procedures for field-collected fire ants should be described.
3. Negative control. In most studies, a negative control should be included. The
number of control replicates should equal the number of replicates for each
treatment. When appropriate, a negative control is typically treated with a placebo
bait, diluent only or receives no treatment at all (i.e., untreated control).
viii. Quality assurance (QA) plan. Protocols should provide for periodic quality assurance
inspections that are adequate to ensure the integrity of the study and consistency with the
provisions of EPA's GLP regulations (40 CFR §160).
b. Data collection and reporting. Study protocols should provide for collection and reporting of
data covering all aspects of the research including those discussed in section (g) of this guideline.
GLP regulations specify that each study protocol should provide for collecting and reporting all
elements provisioned by the GLP regulation at 40 CFR §160.120.
c. Data analysis. Protocols should include a full description and explanation for the statistical
methods proposed to analyze product performance test results, taking into account the specific
study objectives and variables. A statistician may be consulted regarding the sample size vs.
power of the study design and the statistical methods for data analysis when developing test
protocols. Analysis of data is recommended to determine if the mortality rate of the group treated
with the product differs from the negative control mortality and if any within treatment effects
were significant. Ninety-five percent confidence intervals should be reported around any
estimated statistic. Protocols should explicitly describe the statistical model to be used and
demonstrate that the assumptions underlying the model can be met for all proposed analyses.
Restrictions on randomization of any testing components should be clearly documented and
should be correctly accounted for in the statistical analyses. Generally, generalized linear models
(GLMs) are recommended to fit models directly to non-normal (e.g., binomial - which describes
many of the collected product performance data sets) data using an appropriate link function.
GLMs do not involve transforming the response variable, thereby allowing data to remain on the
original scale of measurement. Generalized linear mixed-models (GLMM) may also be
appropriate for correlated data. Software for analysis using GLMs or GLMMs is available in
many widely sold statistical packages. If survival analyses (e.g., Kaplan-Meier Estimator) are
used, justification should be provided for use of the median value to characterize product
performance and demonstrate that the underlying assumptions of these analyses have been met.
One-way analysis of variance (ANOVA) or mixed-effects models can be used if their
assumptions, such as normality, are justified.
(e) Review of protocols for efficacy studies. Protocols proposing novel testing methods (i.e., non-guideline
testing methods) or testing of other species of ants for which the Agency requires data to support product
performance should be submitted to EPA for review before the study begins.
(f) Execution of efficacy studies.
a. Execution of protocol. In cases where a protocol has been submitted to EPA for review, testing
should be initiated when the EPA review is complete and as applicable, EPA comments should be
incorporated into the revised protocol.
b. QA oversight. Product performance testing is subject to GLP regulations at 40 CFR §160. GLP
regulations state that each testing facility should include an independent QA unit. The QA unit
monitors and documents execution of each protocol in accordance with the GLP regulations (40
CFR §160.35). The QAunit should inspect each study at intervals adequate to ensure the
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integrity of the study and maintain written and properly signed records of each periodic
inspection. Please see (b) b. i. above for the discussion of the use of GLP laboratory methods
when conducting pest product performance studies.
c. Protocol amendments. Amendments are planned changes to the protocol and should be made
before the study is executed. All amendments to the protocol should be noted in the written
report to the agency.
d. Deviations from protocol. Even when executing the best-designed and most comprehensive
protocols, unanticipated deviations from the protocol may occur. All such deviations from the
protocol and their impact on the research should be fully reported in the study report submitted to
EPA (40 CFR §160.185).
(g) Reporting of completed efficacy studies to the agency.
a. Study identification. Title, identifying study number(s), sponsor, study director, investigators,
name and location of the testing facility, and dates of the study should be reported. If tests are
conducted outside the U.S., the relevance of the study for U.S. regulatory purposes should be
justified in the study report.
b. Study objective(s). The purpose of the study should be stated, including whether the study is the
laboratory or field component of the data set.
c. Testing conditions. Information on temperature, RH, ambient light and photoperiod (L:D), and
air flow (where applicable) should be reported.
d. Testing system. Testing system information, including but not limited to the following, should
be reported:
i. Identification of field populations, if applicable; where colonies were collected/obtained;
development stage; and methods for preparation of fire ants for testing (e.g., feeding).
ii. Rearing, handling, and maintenance of fire ants.
iii. Description of test substance (i.e., product, % active ingredient, and formulation to be
tested).
iv. Description of the negative control including the application, if applicable.
v. Description of the experimental unit.
vi. Treatment application rate and method of application (rate and method of application
should be consistent with label instructions).
vii. Number of product treatments if greater than one.
viii. Number of replicates per treatment.
ix. Number of individuals or mounds/plots per replicate for each treatment including controls.
x. Length of ant exposure to each treatment.
xi. Endpoints and time intervals of endpoint recordings.
e. Data/results reporting.
i. Raw data. Include legible copies of all raw data.
ii. Results summary. Report summary test results on all aspects of research. The amount of
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product applied, and active ingredient delivered per replicate should be reported. See
sections (i) through (m) for more details.
iii. Data analysis. Provide a copy of the statistical analysis plan and results from statistical
analysis. Refer to Section (d) c. for recommendations on data analyses, unless otherwise
indicated in a study-specific data analysis and reporting section.
f. Study conclusions. The report should include a discussion of the study results and conclusions
based on treatment endpoints. Conclusions should state why and how the study results do or do
not support the tested hypothesis and label claims.
g. Protocol with amendments and study deviations from the protocol. A copy of the study
protocol should be included with amendments and deviations. Deviations should be justified and
described together with their impact on the validity of the study. The study should align with the
protocol.
(h) Retention of records. The record-keeping provisions of 40 CFR §160.190 and §160.195 apply to records
of any study conducted under the GLP rule.
(i) Specific guidance for field studies for testing area-applied pesticide products.
a. Study objective: The field studies described in this section are designed to determine product
performance of area-applied pesticide products, such as, but not limited to, broadcast baits, bait
stations, and products other than baits, against listed fire ants in a colony/mound/nest. In
addition, laboratory tests should be conducted in conjunction with field tests: for baits (see
section (k)); for products other than baits (see section (1)); and for products containing an IGR
(see section (m)).
b. Materials and methods.
i. Site selection. A minimum of two sites should be selected from within the range of the
listed imported fire ants and should not have been exposed to pesticide treatment for at
least one year prior to the study. A site refers to a contiguous stretch of land; therefore, a
minimum of two separated (i.e., non-contiguous) stretches of land should be used for the
study.
ii. Experimental units. The treatment should be applied to a plot within a site. Plots may be
any size, so long as all plots within all sites are the same size, and should contain a
minimum density of active fire ant mounds equivalent to 20 active mounds per acre (no
less than 10 active mounds per plot) to ensure sufficient numbers of foraging fire ants are
present for testing of area-applied treatments (Drees et al. 2013). All plots within a site
should be separated from each other by at least 56 ft or more (Colby et al. 2008, Stringer et
al. 2011). Plots within a site should be as similar as possible with respect to density of
active fire ant mounds, relative numbers of active foragers, and environmental conditions
such as hydrology and vegetation cover.
iii. Number of replicates per treatment. Depending on the results of power vs. sample size
analysis, the study should include a minimum of four plots per treatment per site. The
design should be balanced with an equal number of treated and negative control plots per
site.
iv. Application method. The lowest application rate to be supported on the product label
should be applied using methods consistent with the product label. If bait stations are used,
the number of deployed bait stations and the amount of bait in each station should
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correspond to the lowest number of stations per unit area proposed on the label. Bait station
products should be tested as they would be deployed by a consumer.
Ant exposure to product treatments. Area-applied product studies should be conducted
for a minimum of 60 days post-application at each site. A longer study duration may be
needed based on desired label claims and the product's mode of action.
Data collection and endpoints. All sampling should be conducted when soil temperatures
are between 65 - 97 °F (Porter & Tschinkel 1987, Helms & Vinson 2005, Drees et al.
2007). A pre-treatment sampling of mounds and forager activity should be conducted 1-7
days prior to treatment application. The number of active mounds should be used to
establish homogeneity among plots on a site (see Snedecor and Cochran (1989) for
guidance on randomization of field trials based on number of mounds). Sampling should be
conducted over a minimum of 60 days with a minimum of four sample collections, with the
final sample occurring on the last day (i.e., day 60) or soon thereafter if all samples cannot
be collected on the last day.
The primary endpoint for measuring efficacy of area-applied products in this design
focuses on forager numbers. The number and timing of sampling intervals for foragers
should be based on the pesticide's mode of action and desired label claims. If uncertain
about suitable establishment of plots and/or sampling regime, consult the Agency for
appropriate intervals. Ideally, data should be collected simultaneously from treated and
control plots.
1. Sampling of foragers. The number of foragers present in the plot at each sampling
interval should be estimated. This may be accomplished using an attractive food
lure (e.g., hot dog pieces) and vials. Place a minimum of 9 vials containing the
attractive food lure equidistant from the plot edge and each other within the plot for
an established sampling period, approximately 30 minutes to 1 hour. Vials should be
placed in contact with the soil and provided with some degree of shading from
direct sunlight, such as use of semi-opaque vials (Vogt et al. 2003). Sampling of
foragers may be conducted at 50% of the plot area. At the end of the sampling
period, cap the vials. The number of fire ants in each vial should be counted and
reported for the plot.
2. Number of active mounds. The number of active mounds per plot should be used
to establish homogeneity among plots on a site. A mound is considered active when
at least 20 workers exit the mound immediately following disturbance (Oi & Oi
2006). To determine if mounds on each plot are active, the surface of each mound
should be scratched with a pair of forceps or similar object. Alternatively, a narrow
probe, similar to a stem of a field flag (approximately 0.12"), can be inserted into
the mound from 6"- 8" deep. It is optional to compare numbers of active mounds
pre- and post-treatment for specific mound claims, such as "no new mounds." In
this case, at each sampling interval, all mounds in each plot per site should be
assessed for activity.
3. Insect growth regulators (IGRs). For products containing an IGR, an additional
sampling assessment to confirm the presence of brood in all mounds in each plot
should be conducted. To assess for the presence of brood during a single pre- and
post-treatment sampling of each mound, using a small-headed shovel or hand
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trowel, a small portion of the mound (about the size of a fist) should be turned over
to observe brood. Record whether brood is present or absent in all mounds in each
plot. The post-treatment sample should occur on the last day (i.e., day 60) or soon
thereafter if all samples cannot be collected on the last day.
c. Data analysis and reporting. Refer to Section (g) of this guideline for guidance on data/result
reporting. In addition, the following information should be included in the completed efficacy
studies to the agency:
i. Test Conditions. For each assessment, report the date, time, temperature, and weather
conditions.
ii. Treatment data. The amount of product applied, expressed as weight of product (and
active ingredient) per unit area, should be reported for each replicate. If reapplication or
rebaiting occurred, specify methods and thresholds for reapplication/rebaiting.
For bait stations, the amount of bait per bait station and the duration of baiting events
needed to achieve product efficacy should be specified. The number of bait stations per unit
area and location of bait stations, including distance from the mound/colony and other bait
stations, should be reported. Bait removal by the test species as the difference in pre- and
post-weights and accounting for water loss/gain should be documented.
iii. Forager numbers. For each assessment, the date, time, temperature, and weather
conditions (e.g., precipitation) should be reported. Numbers of fire ants per vial should be
recorded. The statistical analysis should consider the random effect of the plot and the
correlation of data collected from the same plot at different time points. Generalized linear
mixed effects models for Poisson distribution should be used to analyze the number of fire
ants. Estimated count ratio (i.e., mean ratio) between treatment and control and its 95%
confidence interval should be adjusted for baseline values and reported for each time point.
iv. Number of active mounds. For each assessment, the date, time, temperature, and weather
conditions (e.g., precipitation) should be reported. The number of active and inactive
mounds per plot per treatment within a site should be reported for each assessment. If
product application is intended to prevent new mounds from forming on treated plots, then
the number of all active mounds per plot per treatment within a site should be reported for
each assessment. Generalized linear mixed effects models for Poisson distribution should
be used to analyze the number of active mounds, where the random effect of plot and the
correlation of data collected from the same plot at different time points are accounted for in
the model. Estimated count ratio (i.e., mean ratio) between treatment and control and its
95% confidence interval should be adjusted for baseline values and reported for each time
point.
v. Presence/absence of brood if using an IGR. For each assessment, the date, time,
temperature, and weather conditions should be reported. The number of mounds with and
without brood per plot per treatment within a site should be reported for each of the pre-
and post-assessments. If product application is intended to prevent new mounds from
forming on plots with treated mounds, then brood assessments should be conducted on all
new mounds per plot per treatment within a site at each assessment. This establishes the
first or pre-assessment for each of the new mounds identified. Generalized linear mixed
effects models for Poisson distribution should be used to analyze the number of mounds
with brood, where the random effect of plot and correlation of data collected from the same
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plot at different time points are accounted for in the model. Estimated count ratio (i.e.,
mean ratio) between treatment and control and its 95% confidence interval should be
adjusted for baseline values and reported for each time point.
d. Study conclusions. Summarize study outcomes for area applied product testing against imported
fire ants in a colony/mound/nest and discuss their implications for product labeling.
(j) Specific guidance for field studies for testing mound-applied pesticide products.
a. Study objective: The field studies described in this section are designed to determine the
product performance of mound-applied pesticide products, such as but not limited to baits, dusts,
and injectable aerosols, against listed fire ants in a colony/mound/nest. In addition, laboratory
tests should be conducted in conjunction with field tests: for baits (see section (k)); for products
other than baits (see section (1)); and for products containing an IGR (see section (m)).
b. Materials and methods.
i. Site selection. A minimum of two sites should be selected from within the range of listed
imported fire ants and should not have been exposed to pesticide treatment for at least one
year prior to the study. A site refers to a contiguous stretch of land; therefore, a minimum of
two separated (i.e., non-contiguous) stretches of land should be used for the study.
ii. Experimental units. The treatment should be applied to individual mounds on designated
plots within a site. For this field test, a plot is simply the area containing at least 10 active
mounds. Treatment is applied to each active mound within a plot. Plots may be any size.
All plots within a site should be separated from each other by at least 56 ft or more (Colby
et al. 2008, Stringer et al. 2011). Plots within a site should be as similar as possible with
respect to density of active fire ant mounds and environmental conditions such as
hydrology and vegetation cover.
iii. Number of replicates per treatment. Depending on the results of power vs. sample size
analysis, the study should include a minimum of four plots per treatment per site. The
design should be balanced with an equal number of treated and negative control plots per
site.
iv. Application method. The lowest application rate to be supported on the product label
should be applied using methods consistent with the product label. Note: the cumulative
mound application rate should not exceed the maximum label rate per acre.
v. Ant exposure to product treatments. Treatments should be applied no less than 24 hours
after disturbance of the mounds, unless required for product application. Studies testing
mound-applied products should be conducted for a minimum of 30 days at each site. A
longer study duration may be needed based on desired label claims and the product's mode
of action.
vi. Data collection and endpoints. All sampling should be conducted when soil temperatures
are between 65 - 97 °F at a depth of 0.8" (Porter & Tschinkel 1987, Helms & Vinson 2005,
Drees et al. 2007). A pre-treatment assessment of mounds should be conducted 1-7 days
prior to treatment application. Sampling should be conducted over a minimum of 30 days
with a minimum of four sample collections, with the final sample occurring on the last day
(i.e., day 30) or soon thereafter if all samples cannot be collected on the last day. The
number and timing of sampling intervals should be based on the pesticide's mode of action
and desired label claims. If uncertain about a suitable sampling regime, consult the Agency
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for appropriate intervals. Ideally, data should be collected simultaneously from treated and
control plots.
1. Number of active mounds. The number of active mounds per plot is used to
establish homogeneity among plots on a site and to compare numbers of active
mounds pre- and post-treatment (see Snedecor and Cochran (1989) for guidance on
randomization of field trials based on number of mounds). A mound is considered
active when at least 20 workers exit the mound immediately following disturbance
(Oi and Oi 2006). To determine if mounds on each plot are active, the surface of
each mound should be scratched with a pair of forceps or similar object.
Alternatively, a narrow probe, similar to a stem of a field flag (approximately 0.12"
diameter), can be inserted into the mound from 6"- 8" deep. If product application
is intended to prevent new mounds from forming on treated plots, then the number
of all active mounds per plot per treatment within a site should be sampled for each
assessment.
2. Insect Growth Regulators (IGRs). For products containing an IGR, an additional
sampling assessment to confirm the presence of brood in all mounds in each plot
should be conducted and the duration of the study should be extended to a
minimum of 60 days, post-treatment. To assess for the presence of brood during a
single pre- and post-treatment sampling of each mound, using a small-headed
shovel or hand trowel, a small portion of the mound (about the size of a fist) should
be turned over to observe brood. Record whether brood is present or absent in all
mounds in each plot. The post-treatment sample should occur on the last day (i.e.,
day 60) or soon thereafter if all samples cannot be collected on the last day.
c. Data analysis and reporting. Refer to Section (g) of this guideline for guidance on data/result
reporting. In addition, the following information should be included in the completed efficacy
studies to the agency:
i. Test Conditions. For each assessment, report the date, time, temperature, and weather
conditions.
ii. Treatment data. The amount of product applied, expressed as weight of product per unit
area, should be reported for each replicate. If reapplication/rebaiting occurred, specify
methods and thresholds for reapplication/rebaiting. For bait, specify the duration of baiting
events needed to achieve product efficacy.
iii. Number of active mounds. For each assessment, the date, time, temperature, and weather
conditions should be reported. The number of active treated mounds per plot per treatment
within a site should be reported for each assessment. If product application is intended to
prevent new mounds from forming on plots with treated mounds, then the number of all
active mounds per plot per treatment within a site should be reported for each assessment.
Generalized linear mixed effects models for Poisson distribution should be used to analyze
the number of active mounds, where the random effect of plot and the correlation of data
collected from the same plot at different time points are accounted for in the model.
Estimated count ratio (i.e., mean ratio) between treatment and control and its 95%
confidence interval should be adjusted for baseline values and reported for each time point.
iv. Presence/absence of brood if using an IGR. For each assessment, report the date, time,
temperature, and weather conditions. The number of treated mounds with/without brood
per plot per treatment within a site should be reported for each of the pre- and post-
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assessments. If product application is intended to prevent new mounds from forming on
plots with treated mounds, then brood assessments should be conducted on all new mounds
per plot per treatment within a site at each assessment. This establishes the first or pre-
assessment for each of the new mounds identified. Generalized linear mixed effects models
for Poisson distribution should be used to analyze the number of mounds with brood, where
the random effect of plot and the correlation of data collected from the same plot at
different time points are accounted for in the model. Estimated count ratio (i.e., mean ratio)
between treatment and control and its 95% confidence intervals should be adjusted for
baseline values and reported for each time point.
d. Study conclusions. Summarize study outcomes for mound applied product testing against
imported fire ants in a colony/mound/nest and discuss their implications for product labeling.
(k) Specific guidance for laboratory studies for testing bait products.
a. Study objective: The laboratory studies described in this section are designed to determine
product performance of bait products against all life stages of listed fire ants. In addition, field
tests should be conducted in conjunction with laboratory tests: for area-applied products (see
section (i)) and for mound-applied products (see section (j)).
b. Materials and methods.
i. Experimental units. An experimental unit consists of a single test arena containing fire ant
brood, workers, and a queen. The arena should contain a water source for the ants,
harborage for a queen and brood, alternative food source, and the bait to be tested.
Alternate food source and bait should be placed at opposite ends of the arena from each
other and equidistant from the harborage. The test arena should consist of an open-top box
where interior vertical surfaces are coated with a non-stick material (e.g., Fluon® or talcum
powder) to prevent escape of fire ants. Size of the arena (e.g., 15" L x 12" W) may vary but
should allow adequate space for ants to forage. All test arenas used in this study should
have the same dimensions.
ii. Number of replicates per treatment. Depending on the results of power vs. sample size
analysis, a minimum of five replicates per treatment, with a minimum of 100 workers, a
queen, and a recorded and consistent amount of brood per replicate arena, should be tested.
Design should be balanced with an equal number of treated and control arenas. All arenas
should be held under the same environmental conditions. Laboratory testing may be
conducted at ambient temperature and RH in the testing arena as long as conditions are
optimal for foraging behavior, such as, 82° - 91° F (Banks et al. 1981). Conditions that are
too cool or dry will inhibit normal colony activity. In addition, temperature and RH within
the nest cell should also be maintained at conditions optimal for brood development, such
as > 65 % RH and 82° - 91° F (Williams 1989). Also, laboratory colonies should be
provided time to acclimate to the photoperiod, as photoperiod is known to affect foraging
behavior (Lei et al. 2019). Environmental conditions for laboratory testing, including
temperature, humidity, and photoperiod during testing, should be supported by cited
literature.
iii. Application method. Treatment should be product specific and applied in the manner and
duration as directed by the product label. This study design should consist of a choice test
to demonstrate acceptability of the bait product and the alternate food source should be
palatable, established in the literature, and consistent across trials. The alternate food
source should be available ad libitum to the fire ants throughout duration of the study.
Control arenas should receive the alternate food source only.
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iv. Ant exposure to product treatments. Fire ants should be acclimated to the arenas for a
minimum of 72 hours, during which time they are provided with the alternative food source
and water. Dead individuals should be removed prior to treatment. Uneaten food should be
removed and replaced with a fresh food source immediately prior to initiation of the study.
Tests should be conducted for a maximum of 14 days post-treatment.
v. Data collection and endpoints. The total amount of treated bait used, expressed as weight
of product per unit area, should be documented for each replicate. The number of workers
per replicate should be reported. Mortality counts should be conducted at intervals < 48
hours through the duration of the study. Dead individuals should be removed and counted.
Control groups should be assessed in the same manner as those receiving treatment.
Following the final assessment, all nest arenas should be frozen to determine the number of
surviving individuals and for calculation of the total number of workers.
c. Data analysis and reporting. Refer to Section (g) of this guideline for guidance on data/result
reporting. The amount of time for test colonies to acclimate to environmental conditions in the
laboratory prior to the start of the test should be reported. Control mortality should not exceed
25% for workers and there should be no queen mortality. In addition, the following information
should be included in the completed efficacy studies to the agency:
i. Reduction in worker numbers. Mortality counts of workers should be reported and
generalized linear mixed effect models for binomial distribution should be used to analyze
the proportion of number of surviving workers at each time point (= number of surviving
workers/total workers = (total workers - cumulative mortality counts of workers at the time
point)/total workers). The model should take into account the random effect of replicate
and the correlation of data collected from same replicate at different time points. Survival
proportion per treatment group and survival proportion ratio between treatment and control
groups should be calculated with 95% confidence limits per assessment.
ii. Queen mortality. Mortality counts of queen should be reported and mortality rate should
be calculated with 95% confidence limits across replicates per assessment using
generalized linear models or generalized linear mixed effect models for Poisson
distribution.
d. Study conclusions. Summarize study outcomes for bait products against imported fire ant
colonies (i.e., workers and queen) and discuss their implications for product labeling.
(1) Specific guidance for laboratory studies for testing products other than baits.
a. Study objective: The laboratory studies described in this section are designed to determine
product performance of products, other than baits or premises treatments (e.g., soil and mulch
amendments) against all life stages of listed fire ants. In addition, field tests should be conducted
in conjunction with laboratory tests: for area-applied products (see section (i)) and for mound-
applied products (see section (j)).
b. Materials and methods.
i. Experimental units. An experimental unit consists of an individual nest arena containing
fire ant workers, a queen, plus brood affixed to two foraging arenas via plastic tubing (e.g.,
Tygon® tubing) or suitable bridge (Choe & Rust 2008; Knight & Rust 1990). Arenas
should consist of an open-top container where interior vertical surfaces are coated with a
non-stick material to prevent escape of fire ants. The nest arena should contain water and
harborage for the colony. Food should be available in the nest arena during the acclimation
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period, then removed immediately prior to test initiation. One foraging arena should
contain substrate (e.g., sand, soil) treated with the product and the other foraging arena
should contain an equal amount of untreated substrate or substrate treated with diluent-
only. All foraging arenas used in the study should have the same dimensions; the length of
the bridges or tubing connecting nest arena to the foraging arenas should be equal. The
food sources in the foraging arenas should be palatable, established in the literature,
consistent across all trials, and should be available ad libitum to the fire ants through the
duration of the study. Foraging arenas should contain equal amounts of food placed at the
far end of each arena, so ants travel through the substrate. Testing begins when tubes or
bridges from the foraging arenas are connected to the nest arena.
Alternative nest- and foraging-arena designs may be considered if laboratory space is a
concern. Arena design may consist of a larger nest arena (e.g., 24" L x 16" W) within
which two separate foraging arenas (e.g., nursery pots 4" L x 4" W x 3.5" H) may be placed
(Costa et al. 2005). The nest arena should consist of an open-top box where interior vertical
surfaces are coated with a non-stick material to prevent escape of fire ants. The nest arena
should contain water and harborage for the colony. Food should be available in the nest
arenas during the acclimation period, then removed immediately prior to test initiation. The
foraging arenas (e.g., nursery pots) should each be filled with treated or untreated substrate,
each with equal amounts of food provided ad libitum and placed in the center of the
substrate in order to ensure the fire ants cross over the substrate to access food. Foraging
arenas should be placed at equal distances away from the harborage within the nest arena.
Testing begins when the foraging arenas are placed into the nest arena.
Number of replicates per treatment. Depending on the results of power vs. sample size
analysis, a minimum of five replicates per treatment, with a minimum of 100 workers, a
queen, and a recorded and consistent amount of brood per replicate arena, should be tested.
Design should be balanced with an equal number of treated and control arenas. Laboratory
testing may be conducted at ambient temperature and RH in the testing arena as long as
conditions are optimal for foraging behavior, such as, 82° - 91° F (Banks et al. 1981).
Conditions that are too cool or dry will inhibit normal colony activity. In addition,
temperature and RH within the nest cell should also be maintained at conditions optimal for
brood development, such as, > 65 % RH and 82° - 91° F (Williams 1989). Also, laboratory
colonies should be provided time to acclimate to the photoperiod, as photoperiod is known
to affect foraging behavior (Lei et al. 2019). Environmental conditions for laboratory
testing, including temperature, humidity, and photoperiod during testing, should be
supported by cited literature.
Application method. Treatment should be product specific and applied at the lowest
labeled rate and in the manner as directed by the product label. Testing should be conducted
on substrates (e.g., soil) aged to the maximum residual time point specified on the label.
Aging of substrates should occur outdoors or indoors using simulated temperature, rain,
and sunlight. This study should consist of a choice test of two foraging arenas: one
containing treated substrate (e.g., sand, soil or impregnated material) and the other
containing untreated substrate.
Ant exposure to product treatments. Fire ants should be acclimated to the arenas for a
minimum of 72 hours, during which time they are provided with the alternative food source
and water. Uneaten food and dead individuals should be removed prior to treatment.
Foraging arenas should contain a layer of substrate that completely covers the floor of the
arena. The depth of the substrate layer should be consistent and equal in all foraging arenas.
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Testing begins when tubes or bridges from the foraging arenas are connected to the nest
arena. It is important to note that once foraging arenas are attached to the nest arena, ants
may mound the substrate. Therefore, to avoid disrupting colony activity, measures should
be in place during the initial setup that will prevent ants from escaping test arenas. Equal
amounts of food should be available for the ants to forage at the far end of each the
foraging arenas from the entrance. Control colonies should also have access to two
foraging arenas, both containing untreated substrate only. Tests should be conducted for a
maximum of 14 days post-treatment.
v. Data collection and endpoints. The total number of workers per replicate should be
collected. Mortality should be evaluated at intervals <48 hours through the duration of the
study. Dead individuals should be removed and counted. Controls should be assessed in the
same manner as those receiving treatment. Following the final assessment, all nest arenas
should be frozen to determine the number of surviving individuals and for calculation of
the total number of workers per replicate.
c. Data analysis and reporting. Refer to Section (g) of this guideline for guidance on data/result
reporting. The amount of time for test colonies to acclimate to environmental conditions in the
laboratory prior to the start of the test should be reported. Control mortality should not exceed
25% for workers and there should be no queen mortality. In addition, the following information
should be included in the completed efficacy studies to the Agency:
i. Reduction in worker numbers. Mortality counts of workers should be reported and
generalized linear mixed effect models for binomial distribution should be used to analyze
the proportion of number of surviving workers (= number of surviving workers/total
workers = (total workers - cumulative mortality counts of workers at the time point)/total
workers). The model should take into account the random effect of replicate and the
correlation of data collected from the same replicate at different time points. Survival
proportion per treatment group and survival proportion ratio between treatment and control
groups should be calculated with 95% confidence limits per assessment.
ii. Queen mortality. Mortality counts of queen should be reported and mortality rate should
be calculated with 95% confidence limits across replicates per assessment using
generalized linear models or generalized linear mixed effects models for Poisson
distribution.
d. Study conclusions. Summarize study outcomes for tested products (i.e., other than baits) against
imported fire ant colonies (i.e., workers and queen) and discuss their implications for product
labeling.
(m) Specific guidance for laboratory studies for testing IGR products.
a. Study objectives: The laboratory studies described in this section are designed to determine
product performance of IGR products against immature life stages of listed fire ants. In addition,
field tests should be conducted in conjunction with laboratory tests: for area-applied products
(see section (i)) and for mound-applied products (see section (j)).
b. Materials and methods.
i. Experimental units. Guidance for setting up experimental units to test products containing
an IGR may be found in (k) b. i. of this guideline for baits or (1) b. i. for products other than
baits. However, the size of the nest arena should be larger (e.g., 24" L x 16" W) to allow
adequate space for testing the larger colony size. In addition, for an IGR laboratory study,
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harborage in the nest arenas should contain a covered plastic Petri dish measuring 6 in (150
mm) in diameter and filled 0.24 in deep with plaster. After the plaster has set, one or more
access holes (0.04"- 0.08" diameter) should be soldered into the side of the dish, just above
the plaster, to allow access into the nest cell. Transparent yellow acetate paper affixed over
the lid will filter ambient light allowing for observation of brood inside the nest cell with
minimal disturbance (Williams 1990).
ii. Number of replicates per treatment. Depending on the results of a power vs. sample size
analysis, a minimum of five replicates per treatment with 200 - 1,000 workers, a queen, and
a minimum of 0.34 oz of brood per replicate arena should be tested (Banks et al. 1983).
Specific methods establishing larger colonies in the laboratory for extended studies are
explained in Banks et al. 1981. Additional guidance for setting up replicates to test
products containing an IGR may be found in (k) b. ii. of this guideline for baits or (1) b. ii.
for products other than baits.
iii. Application method. Guidance for application of test products can be found in (k) b. iii. of
this guideline for baits or (1) b. iii. for products other than baits.
iv. Ant exposure to product treatments. Fire ants should be acclimated to the nest arenas for
a minimum of 72 hours and provided with food and water prior to being granted access to
the foraging arenas. Tests with IGRs should be conducted for a minimum of 30 days post-
treatment. Additional guidance for ant exposure to products may be found in (k) b. iv. of
this guideline for baits or (1) b. iv. for products other than baits.
v. Data collection and endpoints. For IGRs, data should be collected at intervals > 7days
through the duration of the study. A minimum of three time points for collection of data
should include the following: presence or absence of brood, changes in caste structure
and/or deformities of the brood, or presence of dead brood in the colony midden (trash
pile). If the test product contains additional active ingredients that are not IGRs, then data
collection corresponding to (k) b. v. for baits or (1) b. v. for products other than baits should
be conducted. Controls should be assessed in the same manner as those receiving treatment.
c. Data analysis and reporting. Refer to Section (g) of this guideline for guidance on data/result
reporting. In addition, the following information should be included in the completed efficacy
studies to the agency:
i. Brood. For treated and control replicates, presence or absence of brood, changes in caste
structure and/or deformities of the brood, and any dead brood observed should be reported.
ii. Additional reporting. If the test product contains additional active ingredients that are not
IGRs, then results and data analysis corresponding to (k) c. for baits or (1) c. for products
other than baits should be conducted.
d. Study conclusions. Summarize study outcomes for IGR products against imported fire ant brood
and discuss implications for product labeling.
(n) References.
Banks, W. A., C. S. Lofgren, D. P. Jouvenaz, C. E. Stringer, P. M. Bishop, D. F. Williams, P. D. Wojcik, and
B. M. Glancey. 1981. Techniques for Collecting, Rearing, and Handling red imported fire ants. USDA, SEA,
AAT-S-21.
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Banks, W. A., L. R. Miles, and D. P. Harlan. 1983. The effects of insect growth regulators and their potential
as control agents for red imported fire ants (Hymenoptera: Formicidae). Florida Entomologist 66 (1): 172-
181.
Triplehorn, C.A., N.F. Johnson, D.J. Borror. 2005. Borror and Delong's introduction to the study of insects.
Thompson Brooks/Cole, 7th edition. Belmont, CA.
Choe, D.H. and M.K. Rust. 2008. Horizontal transfer of insecticides in laboratory colonies of the Argentine
ant (Hymenoptera: Formicidae). Journal of Economic Entomology 101:1397-1405.
Colby, D., C. Husseneder and L. Foil. 2008. Microsatellite genotyping of red imported fire ant (Hymenoptera:
Formicidae) colonies reveals that most colonies persist in plowed pastures. Journal of Economic Entomology
101(4): 1062-1067.
Costa, H.S., L. Greenberg, J. Klotz and M.K. Rust. 2005. Response of Argentine ants and red imported fire
ants to permethrin-impregnated plastic strips; Foraging rates, colonization of potted soil, and differential
mortality. Journal of Economic Entomology 98:2089-2094.
Drees, B.M., B. Summerlin and S.B. Vinson. 2007. Foraging activity and temperature relationship for the red
imported fire ant. Southwestern Entomologist 32:149-156.
Drees, B.M., A.A. Calixto, and RR. Nester. 2013. Integrated pest management concepts for red imported fire
ants Solenopsis invicta (Hymenoptera: Formicidae). Insect Science 20:429-438, DOI 10.1111/j. 1744-
7917.2012.01552.x.
Helms, K.R. and S.B. Vinson. 2005. Surface activity of native ants co-occurring with the red imported fire
ant, Solenopsis invicta (Hymenoptera: Formicidae). Journal of the Southwestern Entomologist 30:223-237.
Knight, R.L. and M.K. Rust. 1990. Repellency and efficacy of insecticides against foraging workers in
laboratory colonies of Argentine ants (Hymenoptera: Formicidae). Journal of Economic Entomology 83:1402-
1408.
Lei, Y. Y. Zhou, L. Lu, and Y. He. 2019. Rhythms in foraging behavior and expression patterns of the
foraging gene in Solenopsis invicta (Hymenoptera: Formicidae) in relation to photoperiod. Journal of
Economic Entomology 112: 2923-2930.
Oi, D.H. and F.M. Oi. 2006. Speed of efficacy and delayed toxicity characteristics of fast-acting fire
(Hymenoptera: Formicidae) baits. Journal of Economic Entomology 99(5): 1739-1749.
Porter, S.D. 1992. Frequency and distribution of polygyne fire ants (Hymenoptera: Formicidae) in Florida.
Florida Entomologist. 75:248-257.
Porter, S. D. and W. R. Tschinkel. 1987. Foraging in Solenopsis invicta (Hymenoptera: Formicidae): Effects
of weather and season. Environmental Entomology 16:802-808.
Snedecor, G.W. and W. G. Cochran. 1989. Statistical Methods, 8th ed. Iowa State University Press. Ames, IA
Stringer, L., D. M. Suckling, D.B. Baird, and P.J. Lester. 2011. Sampling Efficacy for the Red Imported Fire
Ant Solenopsis invicta (Hymenoptera: Formicidae). Environmental Entomology. 40:1276-84.
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Torre-Bueno, J.R. 1989. The Torre-Bueno glossary of entomology. The New York Entomological Society.
New York, NY.
Vogt, J.T., W.A. Smith, R.A. Grantham, and W.E. Russell. 2003. Effects of temperature and season on
foraging activity of red imported fire ants (Hymenoptera: Formicidae) in Oklahoma. Environmental
Entomology. 32:447-451.
Williams, D.F. 1989. An improved artificial nest for laboratory rearing of the imported fire ant, Solenopsis
invicta (Hymenoptera: Formicidae). Florida Entomologist 72: 705-707.
Williams, D. F. 1990. Effects of fenoxycarb baits on laboratory colonies of Pharaoh's ant, Monomorium
pharaonis, pp. 676-683. In R. K. Vander Meer [ed.], Applied myrmecology: a world perspective. Westview,
Boulder, CO.
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