Sypport Document #4
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EPA
October 15, 1982
PESTICIDE ASSESSMENT GUIDELINES
SUBDIVISION L
HAZARD EVAUJATIONj NOHTARGET INSECTS
by
Ecological Effect* Branch
Hazard Evaluation Division
Office of Pesticide Program
Guidelines Project Manager
Robert r. Hitch
Hazard Evaluation Division
Office of Pesticide Progr
O.S. Environmental Protection Agency
Office of Pesticides and Toxic Substances
Washington, D.C. 20460
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SubdlTialon L deacrltes protocols which
Tirmt~iirij> L 1 nmi<_L
pectlci
158,
be
BTW*^w4w^^B^ j-»-
i** *A*WU£UETCU| • f^m
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Subdivision L
HAZARD EVALUATION: NONTARGET INSBCTS
Table of Contents
DISCUSSION
I. ORGANIZATION AND PHILOSOPHY OP SUBDIVISION L
II. MAJOR ISSUES
GUIDELINES -.-'• - -
Series 140: SCOPE AND GENERAL REQUIREMENTS
140-1 General Information
140-2 Definitions
140-3 Basic standards for testing
140-4' Reporting and evaluation of results
140-5 Special test requirement*
Series 141: NONTARGET INSECT TESTING - POLLINATORS
141-1 Honey bee acute contact LD50
141-2 Honey bee - toxicity of residues on foliage
144-«3» Wild bees important in alfalfa pollination -
toxicity of residues on foliage
141-4 Honey bee subacute feeding study (reserved)
141-5 Field testing for pollinators
Series 142: NONTARGET INSECT TESTING -
AQUATIC INSECTS
142-1 Acute toxicity to aquatic insects (reserved)
142-2 Aquatic insect life-cycle study (reserved)
142-3 Simulated or actual field testing
for aquatic insects (reserved)
Series 143: NONT1
INSECT TESTING -
PREDATORS AND PARASITES
143-1 Acute and residual toxicity -
terrestrial predators and parasites
vegetable, field/ and cereal crops
(reserved)
143-2 Toxicity to predators and parasites in
fruit and nut crops (reserved)
143-3 Toxicity to predators and parasites -
forest applications (reserved)
in
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1
I. ORGANIZATION AND PHILOSOPHY OF SUBDIVISION L
A. Introduction.
Subdivision L provides guidelines for testing and information
on data submission concerning the effects of pesticides on non-
target insect*. Data developed according to the guidelines in this
subdivision, in conjunction with information obtained through
other subdivisions of the guidelines, will be used to make the
determination, required by FIFRA, as to whether a pesticide will
perform its intended function without causing unreasonable adverse
effects on the environment.
B. Approach
Proposed rule, 40 CFR Part 158, specifies the kind of data and
information that must be submitted to EPA to support the registra-
tion of each pesticide under the Federal Insecticide, Fungicide and
Rodenticide Act. The Agency intends to promulgate Part 158 as a
final role during 1983.' This subdivision provides detailed informa-
tion relating to *^«* data requirements listed in 40 CFR Part 158,
including the conditions under which each data requirement is applic-
able, the standards for acceptable testing, stated with as much
specificity as the current scientific disciplines can provide, and
the information that should be included in a test report.
C. Tier System.
To adhere to the guidelines of this subdivision, the applicant
must submit data obtained from specified toxicity tests. These
tests have been grouped into 3 broad areas: tests for pesticidal
effects on pollinators (f§ 141-1, -2, -3, -4, and -5); tests for
pesticidal effects on aquatic insects (H 142-1, -2, and -3); and
tests for pesticidal effects on insect predators and parasites (§$
143-1, -2, and -3).
Tests for pesticidal effects on pollinators are organized in
a hierarchical, or tier type, system. Generally, the decision as
to whether to proceed to the second tier, or longer term tests,
is based on the potential toxicity demonstrated in the first level
tests (tier I), in conjunction with other pertinent information
such as use pattern and environmental fate profile. Third level
tests (tier III) are designed to provide additional information
with respect to adverse results or conditions reported in lower
tier studies, nnfi are performed under simulated or actual field
conditions. Third level tests will rarely be required, as most
questions concerning pesticide hazard will be answered by the
first and second level tests.
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. Guideline, for aquatic insect temting and for insect predator
and parasite testing have not been developed to date. - •
i are discussed ii
DV Organisation of Section, wimia Subdivision 'i.'.
Within Subdivis ion L , each seetion provides the guidelines
for aparticular test or group of related tests, and contains the
following: a provision stating the circumstances under Hiich data
are required; a provision stating Aether testing mamt be performed
on the •anufacturing-us. product or end-™ product, or both,
visions establishing the ft standard, that must be compSea
in Derating the data, and specific reporting requirement.. m
addition, an applicant must comply with the applicable provisions
I' pfc"1 required- p^-.^.y*. . B^ ^ ^ section, of this sub-
division establishes data reJjuire«nts for a particular test
r est an
begias with a paragraph entitled Ttoen required." tti is paragraph
establishe. the condition, under which data fro. that test are
required to support the registration of a pesticide product"
Virtually every sinufacturing-use product is formulated into
an end-use product the u~ of *ich can pose . risk to non-target
insects, therefore, the first tier studies in Subdivision L (§|
141-1. -3, 142-1.- 143-1, -2, and -3) apply to all «mf,cturiB9-use
products, regardless of eventual intended use, and to all end-use
product, intended for outdoor application, subject £ any exceptions
contained in each section.
2' "Test standards- paragraphs. Specific test staidards for a
study are set forth in the paragraph entitled -Test standards- in
, additi°n' t"t1^ «u«tbe performed in accordance
with the -Basic standards for testing- contained in $ 140-3. me
general and specific test standard, for acceptable titing identify
the factors in th. perfomance of a test that EPA has determined to
be necessary to produce reliable and complete data. The test
standards cover such aspects of testing, methodology as the test
substance, test conditions, test species, age of test org.ni.ms.
and duration of the test.
3* ^Porting requirements . Section 140-4, -Reporting and Evalua-
tion of Results," provide, the general reporting and evaluation
requirements for this subdivision. In addition, each section
contains a paragraph entitled "Reporting of ~d«ta,-'*i* sets forth
In
- — c — — —-y "•»*• *•* <-*** ^ i ±1-1.1 90
the Agency requirements for the data that must be submitted.
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most cases, the minimum data required to support the registration
of a pesticide are described in the first level tests, H -141-1 (c)
and 14 1-3 (c). These data include a determination of the acute
contact XJ350 for honey bees for each active ingredient of the
pesticide, and a determination of the residual toxicity of end-use
pesticide products to alfalfa pollinators.
requirements for aquatic insects and for insect
predators and parasites have not been developed bo date
The Agency intends that the need for data submission required
by ff 141-2, -4 and -5 be relatively infreqoent. However, the Agency
believes that information about these testa should enable applicants
to plan their testing programs sore -effectively.
*• Protocols and reference*. Subdivision L includes paragraphs
at the end of each section that provide examples of acceptable test
protocols and/or reference* that may contain background information
useful in developing acceptable protocols, 3his information is
intended only as guidance to the applicant.
II. MAJOR ISSUES
Agency review of the Subdivision L guidelines has identi-
fied a number of issues Hhich are discussed in the following
paragraphs.
A* Dat* Requirements for Manufacturing-Ose Products.
In the Preamble to the 1978 proposed Guidelines, KPA asked for
public comment on the question whether the data requirements of
this subdivision should be extended to manufacturing-use products.
After serious consideration of this issue, the Agency has concluded
that extending the data requirements to such pesticides is appropriate.
The Agency was Influenced by the views of comment era on this issue
who generally favored a data submission requirement ifeich makes the
basic manufacturer of an active ingredient responsible for providing
t of the environmental fate data.
Therefore, 40 CPR-f. 158.50, entitled "Formula-tors'
requires a registrant of a manufacturing-use product to submit (or
cite) any data pertaining to the safety of an active ingredient In
its product if the same data are required to support the registration
of an end-use product that could Legally be produced from the regis-
trant's manufacturing-use products. (An end-use product is a pesti-
cide product bearing label directions for immediate end-use as a
pesticide). Section 158.50 also provides that such data must be
submitted by an applicant for registration of the end-use product,
except that the producer of the end-use product will generally not
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have to submit or rite data pertaining to registered products
which the end-use producer purchases and uses to formulate the
end-use product. Oils decision reflects the Agency's expectation
that manufacturing-use product registrants will be the major source
of registration data, and that end-use product formula tors will,
in most cases, need to supply much less data. This decision is
consistent with the provisions of, and Congressional intent behind,
sec. 3(c)(2)(D) of FIFRA, which provides that:
No applicant for registration of a pesticide who
proposes to purchase a registered pesticide from
another producer in order to formulate such pur-
chased pesticide into an end-use product shall be
required to—:" ' ' •'' '' •''.'.''"
(1) submit or rite data pertaining to the safety
of such purchased product; or
(ii) offer to pay reasonable compensation otherwise
required by [$ 3(c)(1)(D) of FIFRA] for use of any such
data.
Implicit in sec. 3(c)(2)(D) is Congress' expectation that it
would be the registrant of the manufacturing use product who would
provide significant amounts of data pertaining to the safety of its
product. (See, e.g., Sen. Rep. No. 334, 95th Cong., 1st Sess., pp.
8-9.)
Moreover, if data requirements were imposed solely on regis-
trants of end-use products, sec. 3(c)(2)(D) might be read to prevent
che Agency from obtaining data on the grounds that the data pertain
to the safety of a purchased product.
B. Testing for effects on Terrestrial Insect Predators and Para-
sites. Testing for adverse effects on insect predators and parasites
has been a major topic of discussion and disagreement. Those who
oppose the establishment of predator/parasite data requirements
rite a number of reasons. The regulatory use of such Information
has been questioned on the basis that it is unclear how such infor-
mation can be used in product labeling. Doubt has been expressed
as to the predictive value of pesticide effects data on only a few
major species, when certain agroecosys terns are known to contain
hundreds of species of nontarget insects. Finally, some reviewers
believe that establishment of predator /parasite data requirements
will require an unwarranted expenditure in terms of time and money
for development, submission, and evaluation of the data.
Those who favor the establishment of data requirements for insect
predators and parasites reason that assessment of this hazard is an
inherent part of the Agency's responsibility for overall nontarget
organism hazard evaluation, and that such information is essential
to support the development of strategies under integrated pest man-
agement (IPM) programs. (See also part II.E. of this discussion.)
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If the guidelines should address the hazard to predators
parasite., the question then bee™ one of procedure*^
* V^ to •" ab°Ut making ^^ hazard asses s«n« sveral
approaches have been suggested. »«««/ several
One approach would break down representative nreda to«
be pr*iiitive. It ha. also been suggested, as a »eaz» of reduc-
ing tin. and cost, that only three to four representative
parasite specie, be .elected overall for t«tSg. Such
T0^ r°bably *>•'*<*- ba-ecTo! their a
- and importance in major agricultural crop system.:
Finally, it ha. been proposed that, instead of imposing data
*
, applicants who demonstrate that
product, have utility «ithin IPM sy.t«ms could be :p.StSl
to incorporate po.itive label .tat ement. regarding the absence of
^Sfl! *! w8 °* *"* V*0*™" on nontarget iJects-^e^nc
decision Aether to permit sucH claijns would be based on revieTof
'
* , an
this system would require an amendment pf Subdivision H (Labeling
Requirements for Pesticides and Devices) to pemit
target insect safety claim, on product labelT
C* 'Testing for Effects on Aquatic in.eefc«..
tte sections of Subdivision L that deal with aquatic
insect testing have been designated as reserved. Ihis
was taXen for the following reasons :
1)
Subdivision L requira«nts duplicate those in Subdivision
ment.
Meth0d°logy for testiD9 for pesticide effects on nontarget
especially for
for Tronic
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D. Scope of Testing.
Inherent in guidelines development is the need to strike a
balance between adequate data requirements and increased regulatory
burden. With this in mind, the Agency has considered a number
of potential' data requirements viiioh have not been included in the
draft to date. Generally, the reason for their exclusion is a lack
of methodology for generating the data. Since a decision to include
certain additional requirements would involve development and valida-
tion of methods, the decision mus.t be based on the overall importance
of the particular data requirements.
Specifically, consideration has been given to requiring the
following types of tests: effects of pesticide application
(especially forest uses) on soil/litter arthropods; effects of
systemic pesticides on pollinators and on insect predators and
parasites; effects of mosquito abatedment treatments on pollinators;
effects of pesticide applications on pollinatoiB other than those
already included in this subdivision (s.g. , bumblebees); and
pesticide effects on introduced biological control agents (insects).
z' Relation of Subdivision L to Agency IPM Program.
She Environmental Potection Agency's Integrated Pest Management
(IPM) unit has expressed interest in the development of those Sub-
division L sections which deal with insect predators and parasites. :•.
The IPM unit, has provided suggestions that could serve as the basis ;
for Subdivision L data requirements. Information identified by the
IPM unit as useful includes: ...
- Pesticide efficacy against target pests in an IPM setting;
- Effect on other test species (direct),-
- Effect on biological control agents and other beneficial
organisms found in IPM ecosystems;
- Most appropriate use pattern in an IPM program;
- Probability of resistance developing in pests and beneficial
species; and , -
- Effect on total pesticide use in the system.
These ideas related to "system impacts" rather than specific
effects on selected test organisms. As certain commenters have
pointed out, evaluation of "system impact" involves large-scale,
long-tera studies. Such evaluation would probably be beyond the
limited scope of testing now envisioned for Subdivision L.
Even though it may not be possible to structure Subdivision L
to respond to the broad issues outlined by the IPM unit, it may be
possible to design the predator/pa.rasite testing scheme so that
required data are of value to the IPM unit as well as to the rest
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of the Agency. In other words, issues outlined by tfce 1PM unti
should be kept in mind while the predator/parasite parts of
Subdivision L are being designed.
p- Honey Bee Subacute Feeding Study.
Section 141-4 of Subdivision L has been reserved for a honey
bee subacute feeding study. With the exception of field studies,
the other bee studies in this subdivision are desigaed to assess
pesticide toxicity to individual bees, the importance of the sub-
acute study is that it is designed to assess effects on the colony
as a unit.
A honey bee subacute feeding study is currently being developed
and validated under an SPA contract. The methodology suggested
here and the iss»s noted below were generated during a Hovember
1978, meeting of EPA and DSDA representatives and bee researchers
Participants in the discussion were given the opportunity to comment
on a written summary subsequent to the meeting.
Before data requirements are imposed, there is a need to vali-
date the proposed methodology and to develop clearly defined criteria
for requiring that the study be conducted.
1. Purpose. The purpose of the honey bee subacute feeding study
is to study the effects of low levels of pesticides on honey bee
colonies. The study is intended to identify those pesticides 1*1 icfa
may cause adverse reproductive, behaviorial, or other subacute
effects, and mien can be brought back to the hive because the
foragers exposed to the pesticide axe not killed ootright in the
rield. The adverse potential of such a pesticide would not show up
in an acute toxicity test, nor is it likely that the fairly short
term residual toxicity test would reveal this potential.
2. General approach. The test involves exposure of intact bee
colonies to low levels of pesticide through feeding of the pesticide
i* pollen and in sugar candy or syrup. Through caging or location,
colonies are restricted to feeding only on the treated food provided.
At the same time, control colonies are maintained under the same
conditions, but without exposure to the test pesticide. Periodic
checks on amounts of eggs, sealed brood, ai*l adult population si^e,
^ «8ll1f!,0beer^tl0n8 °f •ortality «* Physiological or behavioral
abnormalities, illow the researcher to de temine whether the low
level of pesticide is adversely affecting the colony.
*I_ —!! required. The Agency believes that requiring the test
wticide to »tich bees might be exposed would not be
terms of the time and money needed for data developi
for every pesticide to *ich bees might be exposed would not be
feasible in *• _«.-«--..
and review. Therefore, the Agency intends to "res tricTtes" ting*" to
int
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pesticides having characteristics which would indicate a
of the pesticide being transported back to the
subacote feeding study would be required to
of each end-use product intended for outdoo
following three criteria apply:
- Honey bee acute contact LD50 testing shows that the LDKn
of any acfciv» j nnr^.n ^»^.» •>_ *.i__ _* . . _ 50
•icrogrj
- Residue
132 o < 1» accordance with
132-2 of Subdivision K shows extended presence of
dislodgeable residues on plant surfaces; and
- The proposed use involve* application to crop, that are
known to be attractive to bees.
Data^frm the study would also be required to support the
ss
JT
5. Dosa«?e range. Ctoe point that bee research experts aoreed ur»n
is that even the highest do.age should not el^S^y £ Se
test colonies during the test period. Beyond that
sue a. a. bases for deterging th'
- The dosages and rates of dosing should simulate field ex-
posure;
- Dosages should be related to exposures corresponding to
reconmended application rates, but should not necessarily
«»1 field ex
field exposure; and
- Dosage range can be roughly determined frc* acute toxicity
data. •*
-all
instances it would lead to outright
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destruction of test colonies. In the case of a known toxic pesticide
even the highest test rate might have to represent some fraction of *
the exposure expected from a recommended application of the pesticide.
However, test rates should be related somehow to exposures
expected from standard applications. In other words, testing might
establish the lowest level at which a pesticide adversely affects
the colony. The researcher must then determine whether this level
of exposure will be attained or exceeded under normal conditions of
Regarding suggestion (3) (above), manipulation of acute toxicity
data might be a valid method for determination of the dosage range.
If this is the case, then the written protocol must provide details
and Instructions as to how this manipulation is conducted.
A final suggestion was that testing should be conducted using
the maximum non-lethal dosage (1C0) rather than a range of dosages.
This would greatly decrease the number of colonies needed for the test.
6* Administration of toxicant. There is agreement that honey bee
colonies will be exposed to the test pesticide through treated food
substances, with the treated pollen and sugar candy/Syrup as the
sols food source. However, there is some question as to when toe
exposure to treated food should begin. Day 1 (day of queen release
or package installation) has been suggested, the rationale being
that this would be the time of greatest uniformity among the test
•colonies." Following this line of reasoning, allowance for any
"waiting period" prior to toxicant administration would result in
loss of the original uniformity among test ixtits, .due to the
differences in rate of development among the colonies.
Those who favor some waiting period prior to toxicant admin-
istration cite several reasons for this. First, they feel that
uniformity among package bee units should not be assumed, due to
normal variation among packages and among qvmens. A waiting period,
with the possible establishment of extra colonies beyond the novtoer
needed for testing, would allow the researcher to select developed
colonies of approximately equal strength. The rationale is that
this would provide for more uniformity among test ixtits prior to
the actual testing. Also, given the, possibility th'at some of the
packages sight fail 03 develop normally, the waiting period would
allow the researcher to ascertain th;is and to replace those failed
units with the "extras" prior to testing. In other Words, pretest
uniformity would be better assured by using this method than by
administration of the toxicant on day one. Another reason cited is
that the bee package is not a true colony until it has had some
time to develop. Thus, exposure to the toxicant from day V would
not provide a valid test of pesticide effect on a colony.
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Those who favor a waiting period to allow for colony develop-
ment suggested time period* ranging from 10-30 days.
7* **«**»? of the colonies. On the subject of feeding the colonies.
two major areas of concern have been identified: determination of
the appropriate food substance to be used, and development of a
system for replacement of food during the test.
_ For the honey bee colony to develop normally, it must have a
supply of protein and a supply of sugar. For the purposes of i-h^
test, there are three different protein sources to choose from ,
and two ways to provide sugar to the colony.
Protein may be provided through feeding of natural pollen,
pollen plus supplement, or pollen substitute. There are arguments
for and against the use of each. For example, a reliable source of
clean natural pollen may be very difficult to find, and the price
might be prohibitive. On the other hand, some scientists question
whether an adequate pollen substitute has been developed to date,
which would allow for normal colony development under the stress 'of
the test conditions. This is another point which must be established
through validation of the methodology.
It has been suggested that the choice of using either sugar
candy or sugar syrup be left up to the researcher. Appropriate
selection would depend to a great degree upon the pesticide being
tested. For example, some pesticides tend to settle to the bottom
of a sugar syrup, which would result in uneven dosage, in such a
case, candy would be the appropriate sugar source.
Pood replacement during the test also presents a number of
problems which can only be worked out through actual testing. For
example, some researchers feel that fresh food should be supplied
on a regular basis, with uneaten food being removed at the same
time. This would mean replenishment of a fresh supply of toxicant
on a regular basis. Others feel that fresh food should be supplied
only as needed, allowing complete consumption of the previous food
supply. The major point of contention here centers around breakdown
of the toxicant over time. Food replenishment on a regular basis
might eliminate toxicant breakdown as a factor in the study. Food
replacement on an "a» needed" basis allows for the toxicant
degradation that, as advocates of this method point out, occurs
under normal field conditions.
Location of food in the hive has presented no problems , the
only suggestion being that food be located on the top bars of the
brood combs.
8* Size and number of colonies. There are two major schools of
thought on the matter of appropriate colony size for t-hiff test.
One group advocates the use of standard hives; the other advocates
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the use of -mini-hives" which may be of wood or styrofoam. Arguments
in support of the use of standard hives are that:
- Use of standard size equipment and colonies represents th«
real world situation; no extrapolation of data is necessary;
and
- Determination of effects on queen (e.g., reproductive capa-
bility) can be made easily within the original hive.
Arguments for use of mini-hives are that they are less expen-
sive and require less physical work; and use of small colonies
under controlled conditions allows for a clearer expression of
adverse effects in a shorter time period.
The first step in resolution of this problem will be to con-
duct the study using both methods. This will provide needed
information on reliability of data obtained, on tiae and manpower
requirements, and on expense. Based on such information, one of
the methods may be recommended or required, or the choice of method
may be at the option of the applicant. In any case, the number
of colonies per treatment level and the number of controls must be
reported, and each number must be no less than five.
9" Cages and testing facilities. Appropriate testing facilities
might be developed indoors, outdoors, under glass, or under plastic.
Wiatever the choice, the following conditions must be met:
Under conditions of the test, it must be shown that con-
trol colonies can survive and reproduce normally; and
Colonies must be maintained under controlled conditions,
and provided with pollen and candy/syrup as the sole food
source.
The major problem which must be addressed is the potential
difficulty of maintaining healthy colonies while, isolating those
colonies, in some way, from any natural food source. Natural
isolation .(i.e., with no type of confinement) is an impossibility
in many areas of the O.S. Caging or other methods of confinement
also present problems, in that the natural behavior of the bees is
severely disrupted. Also, if cages are to be used, the question
arises as to whether the test hives must be caged individually, or
whether all the hives in one treatment can be caged together. It
has been suggested that individual caging of hives presents an ex-
cessive and unnecessary expense; others believe . it - to - be necessary
in order to obtain valid results. This is another point to be
worked out through actual testing.
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10. Observations. The following measurements must be taken at
regular Lnt*r°-»1f (as yet undetermined): area of eggs, open brood,
and sealed brood. Other parameters suggested for evaluation include
gross colony weight, estimated adult population, and amount of honey
storage. Suggested nonquantitative observations include: presence
or absence of disease; discoloration, desiccation, or other abnor-
malities of eggs and larvae; and morphological or behavioral abnor-
malities in adults. It has been suggested that dead bee traps might
be useful in facilitating observation* of abnormalities in brood and
adults.
Suggestions on frequency of observations and on timing of
first observations have been quite varied. Based on suggestions,
measurements as cited above should be taken either once per week,
once every two weeks, or only on days 30 and 60 after queen re-
lease. It can be assumed that the nonquantitative observations
discussed above which involve disruption of the brood nest should
be made on the days when measurements are taken.
Use of the dead bee crap allows daily observation without
disruption of the colony. With regard to timing of the first
observations which involve inspection .of the brood nest, it is
agreed that some <-*-"• should be allowed after queen release for the
colony to become established. Suggested periods range from two to
four days to two weeks. Also, this adjustment period will be
correlated with vfaatever "waiting period" (prior to toxicant
administration) has been decided upon.
One final point concerning observations is evaluation of effects
on the queen bee. It has been suggested that measuring sealed brood
is an adequate measure of effect on the queen's reproductive ability.
This might suffice in a test run with standard size hives. If mini-
hives are used, this evaluation will involve placement of the queen
in a healthy standard colony at the end of the test psriod.
11. • Pesticide analysis. It has been suggested that pesticide
analysis be performed on the treated and control food mixes (as a
minimum) , and possibly on wax, honey, and dead be«a. Appropriate
timing and frequency of analysis may depend on a number of factors.
12. Disease control. Disease control in the test colonies can be
looked at from two points of view. On the one hznd. it say be
desirable to make preventative treatments to control the cannon
bee diseases *faich might interfere with evaluation of pesticide
effects. On the other hand, it has been suggested that no such
treatments be made, and that disease levels simply be monitored.
13. Minor issues. The following are brief discussions of several
minor issues which have not been resolved:
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of nrrnlpeint. To avoid «nnfr^BriMH«n_ ^fc K.g
suggested that all hive equipment be destroyed after a teat A
question has been raiaed as to whether this is necessary, or whether
some Method of :rtenon>.aeIimfejon -could be-emnloved to aiirw *._.,.. ~*
the hive equipment. Toe answer to thia Ss2£i conl^ST I^Sof
factor in the decision isiether to use a mini-hive for testing.
Destruction and replacement of mini-hive equipment would not be
unreasonably expensive, while regular replacement of standard hive
equipment prior to each test might not be feasible froa a cost
viewpoint.
(b) water supolr. «hen eircnmmtances require tliat water be
supplied to bee colonies, tfais is usually done by proridlng «ter
near tne nivrn enrrance or inside the bive. Such prorision nas
been suggested as appropriate for tnis test. However, anotber
suggestion i* that the water supply should be located ontaide the
hive and off the ground, forcing the bees to fly. -^
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SUBDIVISION L — HAZARD EVALUATION: NONTARGBT INSECTS
Series 140: . SCOPE AND GENERAL REQUIREMENTS
§140-1 General information.
(a) Scope. This subdivision addresses the potential adverse
effects of pesticides on three categories of nontarget insects:
pollinators; aquatic insects; and predators and parasites. Sec-
tions 141-1 through 143-3 of this subdivision provide detailed
information relating to the data, on toxicity to terres trial and
aquatic nontarget insects Wiich are required by 40 CPR § 158.155
to support the registration of a pesticide product. Bach section
specifies the conditions under Waich specific data are required.
In addition, each section contains standards for acceptable testing
(test standards) and a discussion on reporting and evaluation of
data. Finally, many of these sections contain suggested protocols,
references to protocols, or both.
(b) Application of requirements. (1) "When required" and
"test substance- requirements. The registration applicant should
be careful to distinguish between the "When required" and the
"test substance" paragraphs of each section of this subdivision:
(i) The "*ien required" paragraphs pertain to the circum-
stances under Wiich data shall be required by 40 CFR § 158.155,
and specif^' the, .categories of products for which data must be
generated to support registration applications. The test data are
ordinarily required to support the registration of each end-use
product with the prescribed use pattern and each manufacturing-use
product used to make such an end-use product.
(ii) The "test substance" paragraphs refer to )d.nds of testing
required to produce acceptable data, and state the kind of pesticide
material that must be used in each test. The test substance for
studies in this subdivision may be the technical chemical, a typical
end-use product, or an end-use product representative of each
different type of formulation that would be used where pollinating
insects might be affected. Generally, each of these test substances
is prepared by the basic manufacturer of a pesticide chemical.
(2) Testing to meet requirements. Since studies required
by 40 CFR $ 158.155 would ordinarily be conducted by the basic
manufacturer, pesticide formula tors would not often be expected to
conduct such tests themselves to develop data to support their indi-
vidual products. They may do so if they wish, but they may also
merely rely on the data already developed by the basic pesticide
manufacturer.
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IS
system. *„«* fall ^ ^
=r=5=SI Si'JT
£££fL££: tt" —". to*ici—- t.
(«) Relation of-CFR 40. Paa-t I^B ^« e^w^^—j j
indicate clearly each product and
--ta
for th. relation of tne product to
§140-2 Definitions.
(a) The tara "insect," as
the member* of the cLass Insecta
J»eaber» of the class Arachnida,
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16
(d) The term "typical end-use product" means a pesticide
product that is representative of a major formulation calory
(e.g., emulsifiable concentrate, granular product, wettable powder)
^contains the active ingredient of the registration applied*.'
$ 140-3 Basic standards for testing.
The standards contained in this section apply to all studies
in this subdivision unless another section of this subdivision
contains a specific standard on the same subject. In such a case,
the specific stairiard shall apply. t*»«»
(a) Test methods. |1) Toxici ty tests should be conducted
according to uniform methods, *enever possible, to maximize thf
number of reliable comparison, that can be made concerning relative
toxirity and relative sensitivity. ""^relative
Tests should include concurrent control groups to determine
(2)
of
(3) Field tests presenting data in terms of reduction (or
nonreduction) of numbers of nontarget insects should be desired to
include pre-applicatlon counts as well as post-application counts.
^ (b) Test substance. (1) Sections 141-1 through 143-3 of
this subdivision specify whether the data submitted in support of
an application for registration should be derived from tests con-
ducted with the technical grade of the active ingredient or the
end-use product, or both.
' (2) The technical grade of the active ingredient is commonly
the same substance a. the manufacturing-use product for *ich regis-
tration is sougnt or which is used to produce the end-use pesticide
product for *ich registration is sought. In this case JJer.
these guidelines require tes ting of the technical grade of the
bettelted9redieilt' * 8a*ple °* the »*n<**»cturing-u«e .product shall
(3) Some sections require testing with a typical e«d-use
product.
(4) In addition to or in LLeu of testing otherwise discussed
" subdivision, the Agency may require testing to be conducted
(i) An analytically pure grade of an active ingredient;
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17
(11) tte technical grade of an active Ingredient;
(ill) tte inert ingredlent(s) of a pesticide fbnmlation;
(iT) A eonl safimit or disparity of an active or inert ingre-
dient;
(v) A plant or anisal eetabolite or degradation product of
an active or inert Ingredient;
(vi) The pesticide fbzstulation;
(vii) Any addLtioaal substance which could act as a syaergist
to the product Cor which registration Is sought; or
(vili) Any "•—>•«~-»"i~. of substances in paragrapha (b)(4)(i)
through (vii) of this section.
(5) Ihe teat snbatanoe aball be within tiie Halts, if any,
certified in accordance with the reqaireeents of § 62-2 of Sabdivia ion
D - Chiailiil i j KaqtELreeenta: Piuduut: Hueilil i j Bie coapoaition of
the teat sobatance shoqjfl be deteradLned, incladLng the naee and
tieB of known contaadnanta and ijeporitiea, ao far aa is technically
feasible, the '*»»-»^*^"*•<*» should also inclnoa qrauititiea of
onknown materlaLa, if any, ao that 100 percent of the teat
ited for.
(6) the lot and aavple ntuebers of t*"» test snbatance ebotild
be determined 'and recorded. • • •
(7) The teat snbatance sbould be stored under condition that
•«1ntvt1n its stability.
(8) If a carrier or vehicle is need to dissolve or dilate
the test substance, it should be chosen to possess as eany of the
following characteristics as possible:
(i) It should not interfere with absorption, distribution,
•etabolise, or retention of the test substance;
(11) It should not alter the cheaical properties of the test
substance and not enhance, reduce, or alter the toxic characteristics
teat «"*^^»*««~»j
(ill) It should not affect the food and water consnption of
the test Inserts; and
(iv) At the levels used in the study, it should not produce
physiological effects or have local or systealc toxidty in insects.
In addition, such a carrier or vehicle should, if possible, closely
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18
resemble, as to solvent polarity, the carrier or vehicle to be used
xpected conditions of use.
(c) Care and selection of test insects. (1) All data
submitted in. support of an application for registration should be
derived from tests conducted in accordance with good laboratory or
field practices for handling and caring for test insects. Only
healthy insects should be used, and they should be kept in condi-
tions conforming to proper cultural practices.
(2) Insects selected for testing should be common or
representative species currently established in the United States;
they may be laboratory-reared or field-collected.
(3) Insects should be randomly assigned to test groups to
« -in •)••!«. bias and assure comparability of pertinent variables.
(4) "Hie number of insects tasted per concentration and the
of concentrations or dosage levels evaluated should be suffi-
cient to yield statistically sound data.
(5) The insects in each test should, as nearly as practicable,
be of uniform size, age, and sex.
(6) In no circumstances shall threatened or endangered
species be used as test organisms.
(d) Observations. Observations should be made as frequently
as necessary to record visible signs of toxidty and abnormal
behavior.
§ 14C^4 Reporting and evaluation of results.
Each test report submitted under this subdivision should sa-
tisfy the recommendations for reporting and evaluation of data in
this section, unless a specific section elsewhere in this subdivi-
sion directs otherwise. The test report should include all infor-
mation necessary to provide a complete and accurate description of
test procedures »*** evaluation of test results. The test report
should include a summary of the data, an analysis of the data, and
a statement of the conclusions drawn from the analysis. The summary
should be sufficiently detailed to permit the reader to understand
independently the conclusions of the author. Data should be reported
using the metric system.
The test report should include the following information:
(a) Test method. (1) Statement of test method used and a
full description of the experimental design and procedures;
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19
(2) The length and actual dates of the stody;
(3) Ihe name and address of the laboratory performing the
test, and the location where the test was performed; and
(4) Tne name(s) of the principal investigatorts).
(b) Test substance. (1) Identification of the test sub-
stance, including chemical nave, and
(2) Manufacturer and lot and sample numbers of the test
substance.
(c) Test insects. (1) Identification of test insects
(scientific names);
(2) Rationale for selection of spades, if spades used is
other than that specified or preferred in this subdivision;
(3) Age, sex, developmental stage, sice, and weights of test
insects, as applicable;
(4) Source of supply of the insects;
(5) Strain or colony desigaation of the test insects, if
appropriate;
(6) Method used in assigning test insects to test and control
groups; and
(7) Description of any pretest conditioning, including diet.
(d) Dosing or treatment. (1) Description of method, route,
and frequency of administration of test material;
(2) Rationale for selection of method, route, or frequency
of administration, if it is different from that recommended in this
subdivis ion;
(3) Total volume of material administered (test substance
plus carrier);
(4) Identification of any diluents or other materials used
in administering the test substance;
(5) Concentrations of test substance(s) administered (i.e.,
micro grams of test substance per milligram of body weight of the
insect [or micrograms of test substance per test insect] or parts
per million of the test substance in substrate, medium, or water)
or application rates of test subatance(s) expressed as pounds of
active ingredient per acre and kilograms of active ingredient per
hectare;
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and
20
(6) Description of the doe ing or treatment of control insects;
test ^BTibstanee is administered in the diet or
~™ ' !•"»*• A*J wv*. ou Xu U1C u^.6 C OF
water, the assay method used to determine the concentrations of th,
test substance.
(e) Observations. (1) Frequency, duration, and method of
observation;
(2) Detailed description of the nature, incidence, tiae of
occurrence, severity, and duration of all observed toxic effects,
including death aad any other abnomal or unusml signs aad
sympt
(f) Environmental conditions. (1) Terrestrial species. A
description of the rearing conditions during and prior to the test
including: , ," '
(i) Ambient temperature and humidity;
(ii) Photoperiod and lifting;
(iii) A description of the diet, including identification
and/or composition and sources of diet; and - .
(iv) Source of water.
(9> Data analysis. (1) Tabulation of the respans* data at
each treatment level;
(2) Calcuation of the LD^ , and the 95 percent confidence
intervals **ien sufficient doses and test organisms are used to
establish a dose-response line;
(3) Methods of calculation;
(4) No observed effect level; and
(5) Statistical methods used for analysis of data.
(h) References . Complete reference to any published literat-jr«
and copy of any unpublished Literature used in developing the test
protocol, performing the testing, malting and interpreting observations,
or compiling aud evaluating the results should be submitted.
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21
§ 140-5 Special test requirement*.
Series 141: BOH
5 141~1 Honey bee acute contact LPSO
(a.) iteen required. (1) tod-usa arcxfaet.. Data on the acute
Se^Si^M* S050' «« 5?9nired by 40 CFR § 158.155 to support
the ristr.tlon of e«* ^^Trodoct intended for
2) """g'ctnrigq-Me nrodnefc.. Data on the acute contact
(MSO> •» required by 40 CFR $ 158.155 to supporTST
h1*CUra9^e ^"^"^ *** «« legally
and hsicall b
a
be used to p«du« an end-use product subject to
tb. data requirement in paragraph (a)(1) of this section.
(3) See 40 CTR f 158.50, -FoaulabDrs' Exemption,- to deter-
mine whether th«. data .u.t be submitted. Section n-k of this sub-
division provide. « .Iditional discussion on this subject.
(b) Test standards. In addition to satisfying the general
sSl3^?* C°nt*ln*d in 5 140-3, a honey bee «ute cStact u>50
study should meet the following standards:
. M) Subs trace to be tested. The technical grade of each
active ingredient in the product shall be tested;
«. Testing shall be performed on the honey bee
Apis nellifera L.;
(3)
Age. Test insects should be worker bees of mifora age.
(c) Reporting of data. Information to be reported should
<*et the general reporting guidelines of * 14(^4, except that in
cases *ere the LD50 will be in excess of 25 micrograZ/bee . no^
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22
li.it,
Atkins, E.L., Jr., L.D. Anderson/and T.O. Tuft. 1954
—— and technique used in laboratory evaluation of
Atkina, B.L., B.A. Grey^ood, and R.L. Macdonald. 1975
Toxidty of pesticide and other agricultural AemicaU to honey
tadie-
5 141~2 Honey Bee - Toxicibv of Residue* on
(a) ihen reenired. (1) End-uae prod^*.. teta on reBidnal
f^"^1""11011"3 ** 4° CFR § 158'155 to •
of ea* end-u« product intended for outdoor
Ji) «ien the proposed use pattern indicates th*t honey bee.
«ay be exposed to the pesticide; and ^
(ii) tfcen the feaulation contains one or .ore «:tive
ingredients having an acute contact U>50 of less than 11 micrograw/bae,
„ ^ . ^, --- 4^,., rmrLclLji
to bee. are required by 40 CFR f 158.155 to support the
of each manufacturing^,, product -hich can legally and
be used to produce a fomulated product subject to the *tl
ment in paragraph (a)(1) of this section.
(3) See 40 CTR § 158.50, -Foniulators' Exeaption • to
determine whether these data .us t be submitted. Sectioa II- ^
this subdivision provide, an additional discussion on this subject
(b) Test standards. In addition to satisfying the general
LV°ntalned in § 14Q'3' '»*• StUdy 8hOQld — t' the
standards:
'. . (,1) S^tance to be tested, tte test substance shall be a
typical end-use product.
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23
Zf an applicant's product is an end-use product, the
test substance shall be a product isiose femulation is typical ol
the foxsnlation category (e.g., wet table powder, enulsifiable eoi
centrate, granular product) to *iieh the prodnct belc
applicant's product is a •mnafacturing-nae prodnct
the test substance shall be a prodnct representative of a aa^or for-'
itegory wale* Inrlnrtns that end-ns* product, if the
' •
.ly mxnnlatad Into
or ««• «-« fenmla , a •4QUab. .tndy
,
sbcmld be psrfoiMd with a typical end-ue prodnct for
catenary.
(2) Speciea. Vesting shall be pezfomwd on the hoi
Apia nelllfen L.
(3) Age. Vest iaseets shoold be vozknr
of
(4) g»Peri»ental deaion. A. ranflf»lT«rt block deilga shoald
be need in assigning field plots.
(5) B««ldae analysis. Becidne analysis of foliage
old be
*• *• tasting, in accordance with § 132-2
of Subdivision C.
(6) *PI>Hcatian rates. Ihe test substance should be applied
at the proposed label rata(s).
(c) Reporting of data. In addition to the information
specified la f 140^4 of this subdivision, the test report should
contain the following information, to be obtained at the
pesticide application and for the duration of the study
(1)
Weather conditions during and after application;
(2) Ancrant and type of precipitation during reaidue
period; and
(3) Tenpexatnre and humidity data for the period following
application. ,
(d) Acceptable protocol, the following is an euxple of
an acceptable protocol:
Introduction
the purpose of this test is to •easure the
tOKicity of field-weattiered pesticide residiKs
to honey bees. Bie foianlation is applied at
proposed label rates and the reaiduea are
allowed to weather under natural conditions.
At specific periods of tine following.application.
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24
treated foliage is collected and bees are con-
fined on the foliage. This allow for da termin-
ation of the duration of residual toxicity of
a pesticide to honey bees. To reduce the
/jrariability inherent in any type of field
testing, the following suggestions are made:
(1) Weather conditions should be carefully
/monitored during the testing (precipitation
and temperature are two extremely important
factors in the breakdown of pesticide residues);
, (2) Tests should be replicated over time
to reduce variability due to weather conditions;
(3) Test bees should be obtained from
several different colonies/ as bees from
certain colonies may esdiibit increased1 tolerance
to some pesticides.
Materials and Methods
Test crop. The preferred test crop is
alfalfa; an extensive amount of residual
toxicity tseting has been conducted using this
crop. Another crop may be substituted, however,
in situations tiiere alfalfa may not be feasible
or appropriate.
Plots. Small plots (suggest 0.01 acre)
should be designated In alfalfa which has been
grown according to standard agronomic practices.
As noted above, a randomized block design
should be employed for the crop.
Pesticide application. The formulated
product should be applied at the maximum
proposed rate. Lower rates may be tested at
the discretion of the registrant.
Teat residues . Residues should be
allowed to weather in the field for a specific
time prior to collection of foliage samples
for testing. For purposes of comparison, test
samples could be collected 3, 8, and 24 hours
after application. If mortality of bees
exposed to 24-hour-old residues is greater
than 25 percent, sampling at 24-hour intervals
should continue until mortality of bees exposed
to the treated foliage is not significantly
greater than control mortality. Foliage samples
should be chopped and mixed prior to introduction
of bees. Approximately 500 cc of treated
foliage should be placed in each cage.
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25
b SS2SE' Si»ple cages are constructed with
top. and bottoms of 150x15 mm plastic pstri
MS. wire screen is cut into a strip 18x2
f*^!5 sSrrtSfs^'ffi'if-
bottcsi of the cage.
Feeding. Bees should he fed during test-
ing by providing cotton squares (2x2 in or 5x5 c«)
soaked with 50 percent sugar syrup and placed under
the treated foliage.
Treatment and testing of bees. Worker bees
should be collected frost the Craves of established
colonies. Bees may be anesthetized with CO,, if
necessary, prior to their introduction into test
Fifty to 100 bees should be introduced into
each cage. Bees should be caged with the treated
foliage and the cages held at 75-78-F (24-25.6-C)
during the test period.
Mortality should be determined after 24
hours of exposure to the treated foliag*.
Bi test shall consist of at least three
cages of bees per replicate, and each treatment, in-
cluding controls, shall be replicated at least three
tiaes. ":< ' • .
References, tte suggested protocol was developed fro.
the following references, which »ay provide additional information:
and
. , * resear* inv^tigations, 1977. Dcpt. of
St. Oniv. Dnpubl. 22 pp. *"=*»•• «t
(2) Lagier, R.F., c.A. Jchansen, M.G. Kleinschmidt , L.I.
'teD°n0a*' 4nd S'D- J-cteon. 1974. Adjuvants decrease
co11- of
141~3 »^d bee, important in alfalfa pollinati^
residues on foliage. :
toxiclt* ar^" raqUifedt (1) gnd-use product. Data on residual '
_.» __ , . re^uir by 40 CFR § 158.155 to support the registration
Co alfalfa
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26
(2)
toxicity
- _. * " * .-».,.,., to support the registration of
each manufacturing-use product which can be legally and physically
used to produce an end-us* product subject to the data reaiiremenJ
in paragraph (a) (1) of this section. '-juo-temenc
(3) See 40. CFR § 158.50, -Formulators' exemption •
whether these data must be submitted. Section II-A of th
vision provides an additional discussion on this subject.
^ (b) Test standards. In addition to the general test standard.
contained in § 140-3. this study should meet the following standard^
• . (,1' 5ub«t*nce to be tested. The test substance shall be a
typical end-use product.
' (i) If an applicant's product is an end-use product, the test
substance shall be a product whose formulation i. typicafof the
formulation category (e.g., wettable powder, emulsifiable concentrate
granular product) to which the product belongs. concentrate,
(ii) If the applicant's product is a manufacturing-use product
the test substance shall be a product representative of a major
f!^1!!10? category ^ich includes that end-use product, if the
manufacturing-use product is usually formulated into end-use products
»^S fif!i!1?-0r ^rC Mj°r for»ul«tion categories, a separate
study should be performed with a typical end-use product for each
such category.
(2) Teat crop. Alfalfa should be used as the test crop.
(3) Species. Testing shall be performed on the alfalfa
leffCftln9 ***' Meqachile rotunda, and the alkali bee, Nomia
mexanoeri« • . • • —"
(C) ^Portin of data. Information to be reported should meet
°f § 14°-4 "d **
* *< of this test is to measure the toxicity
of field-weathered pesticide residues to alfalfa leaf-
cutting bees and alkali bees, as these species are
important in the pollination of seed alfalfa. The
methodology to be used is the same as that used for
testing residual toxicity to honey bees ({ 141-2 of
this subdivision) with the following exceptions-
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27
M) ctge^* leafCUttlll9 bee: o»e 20-40 bee. p.r
per cage,
(2) AlJoli bee: use 15-30 be«
(«) Reference
following
of inBeeticldea bo
Sta., Coll. of Aqric.. W.h. e^~~^11^"^!^ °^1_ **"h- A^ic. Kxp.
pp. . St.
5
5 U1~5
=
s£s Sss.-st-a ssr
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28
-'-•sss#s^^rur?irrrL«ssr
bo aararrr jr^h /. > / •« \ _« ._,_ ^ _ . ptoauccs
. c. u»rt to bra '
Sane acceptable protocol* My be developed fro. the following
references:
Atkins, E.L. , Jr., L.D
Series 142: NONTAROST INSECT TESTING - AQUATIC INSECTS
5 142~1 *5Ut* to^eifcv to aauatic inaeefca. (reserved)
5 U2"2 ^?^ati= iaaect life-evel^ a^,Hy. (reserved)
§ 142-3
Series 143: HONTAHGET INSECT TESTIHG - PREDATORS AND PARASITES
or actual field ....inq for acTM,ir
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