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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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                                   8


  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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                                    18
       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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                                   11

  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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                                 12

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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                                   13

                 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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                                    14

         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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