#3
5PA-S40/9-82-020
er
J
ana
Thomas C. £lUvangtf
of PwticJrie Pf
Robert K. Hitcn
Haiird Evaluation Di
a of ^estts.da ^
U.S. E
1 a 3*
Aaah-.gts- - C *
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Foreword
Subdivision J describes study protocols **iich may be used to per-
form phytotoxieity testing to support the registration of pesticides
under the Federal Insecticide, Fungicide, and Rodenticide Act (P1FRA).
Public comment on subdivision J was accepted in a series of public
meetings the last of **sich was held in July, 1982. Data requirements
established by 40 CFR Part 158 are discussed in Subdivision J so that it
can be read as a complete package and so that the.protocols can be
be explained in their proper context. ,
J
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SPA
October 27, 1982
PESTICIDE ASSESSMENT GUIDELINES
SUBDIVISION J
HAZARD EVALUATION:
NONTABGET PLANTS
by
Robert w. Hoist, Ph.D.
and
Thomas C. Ellwanger, Ph,.o..
Office of Pesticide Programs
Guidelines 'Projects Manager
Robert K. Sitch
Hazard Evaluation Division
Office of Pesticide Programs
U.S. Environmental Protection Agency
Office of Pesticide and Toxic Substances
Washington, D.C. 20460
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"~\ Subdivision J - Hazard Evaluation: Nontarget Plants
•. ' • ' Table of Contents
DISCUSSION
I. Introduction . 1
II. Organization 2
III. Major Issues . 3
GUIDELINES
Series 120: GENERAL
§ 120-1 Overview ' . 14
I 120-2 Definitions 17
§ 120-3 Basic test standards 19
§ 120-4. General evaluation and reporting requirements 22
Series 121: TARGET AREA TESTING
§ 121-1 Target area pJiytotoxicity testing 28
Series 122: TIER 1 OF NONTARGET AREA TESTING
&••:';. 4 § 122-1 Seed germination/seedling emergence and
*»»"-** vegetative vigor (Tier 1) 38
§ 122-2 Growth and reproduction of aquatic plants
{Tier 1) 40
f 122-30 Acceptable methods and references 42
Series 123: TIER 2 OF NONTARGET AREA TESTING
§ 123-1 Seed geraination/seedling emergence and
vegetative vigor (Tier 2) •. 48
| 123-2 Growth and reproduction of aquatic plants
(Tier 2) 49
Series 124: TIER 3. OF NONTAHGET AREA TESTING
| 124-1 Terrestrial field testing , 51
f 124-2 Aquatic field testing • 53
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1
SUBDIVISION J — HAZARD EVALUATION! MONTMGET PLANTS
DISCUSSION
I. Introduction
The performance requirements and testing and reporting
tores of pesticide, chemical, environmental, Ind toxiclty p*
Tin &ftl*r*f+9'+' **Vk4t *.-,—..' _*_: j_ s . . • *"»**"•**»£ jrf4.
r0n ° m&Ch ***^<*** ***** the Federa
Ptm9lcide' •»* 3°denticide Act
Cod /?!T^ SerleS* ThC firSt iS V°luae 40
Sd informtt'f f*?*^"* ^^ ^^ specifies the kind oata
and information that must be smtmitted. Section 158.150 specifies
testSf °XSn.r^UiT"IMt- f°r Ph^otox^^y .(plant protection)
final yteRdS ^ rOBUl
. the second series of documents [Guideline Subdivisions such as
the present one, published by the National Technical Int option
SrS iS? !] Pr°:iSie ^ tSSt °riteria ^ ^^rting pr^eSes
-
ano data recrement e
SitS i ^ »t««i»rds and protocols for acceptable testing,
stated with ;a« m«ch specificity as the current scientific -di.ci
P^« -llo., :«d reporting procedures. Also provided in Sis
"Mi^"- «• circumstances under which an applicant shoSd
consult with the Agency before initiating a «udy. '
are pri P*?t*ct**m test «««oool. and reporting procedures
r*^1^ ^^ wblll««d alon5 with data on env.ron-
l TCY *" '"^ to «"•*• '^- Potential hazard
on nontarget plants, both terrestrial and amiatic
Cr°PS' "»— «:1.., «d others Sat «i
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A parpose common to all tests i. to provide data which will be
used to determine the need for (and support the «ordLg for) pre-
cautionary ^labeling or other statements^ minimize^ pSinSl
*"«**V** *^*' Generally, the regnant will
^^ Precaution«y Deling with respect to nontaget
such as crop.,, ornamentals, and the like. However, there
may be Situations where the Agency will have to develop additional
precautionary labeling. For example, the spraying of herbicides
may 'not be permitted in the vicinity of critical habitats "of
II. Organization
ad*.. *^ continues with presentation of the major issues
addressed by ccmmenter, with the publication of the proposed guide-
lines - subpart j, Hazard Bvaluation: Nontarget Plants and M
( " ^ ^948.72978 ,
The Guidelines portion ;of - this subdivision (p. 11} is
•
,20
the subdivision (J 120-2) , basic standard, for testing (§
and the general evaluation and reporting procedures^? 120-4).
1"1 d**la'With' *«?•* «** phytotoxiclty testing,
is used to evaluate pesticide toxicity to those plants that
would experience intentional -application . •
™.<« ' series (Series 122, 123, and 1245 com-
prise the tier .testing sequences (Tiers 1, 2, and 3, respectively)
employed to study and report on pesticide toxicity to nontarget
HL? l\ ^ •"•«•.<*•-<*» P*aticid.s are determined through
a series. of, test* as. dictated by specific requirements of each
w««t and tier. The tests are designed to provide guidance for
gathering pesticide effects information on terrestrial and aouatic
plant growth and 'development.; The influences of geographical," sea-
sonal, and species variation are also addressed.
Also contained in a section la Series 122 are detailed proto-
cols for .some of the- studies found in Subdivision J. At' the end
of each protocol are selected references to acceptable methods
that may be used to develop 'pesticide .ohyto toxicity data.
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.,. _ j
Each teat section contains an opening paragraph restating the
circumstance's and for what products, as found in 40 CF.R .Part 15.8,
the data. are. required. The test sections also contain specific -
test criteria, procedures and reporting formats which, in addition
to the respective general testing information, apply to the accom-
plishment of the studies.
The execution of studies in the higher tiers depends on the
results of studies in the lower tiers . The tier system is intended
to reduce repetitive- consultation between the registrant and the
Agency about the need for tests of greater complexity* As a result,
the time required to develop .data. for registration of a pesticide
should be reduced substantially. - • '. .
III. MAJOR ISStJES
The Agency received comments from numerous persons or groups
regarding the :1980 proposed guidelines and the 1982 draft of this
document. In many cases the cosaaenters provided information on
the applicability and the scientific merit of the various . tests.
In response to these public .comments, -the Agency has modified or
clarified all sections and many paragraphs of these guidelines.
Only the more significant and controversial issues submitted by
the public are discussed in the following pages. Many recommen-
dations were adopted by the Agency which do not wax-rant discussion
here. . . . ' -
' A. General Information .
Several commenters have expressed concern that the Agency,
through proposed Subpart J and the 'other proposed subparts, is
trying to investigate whether all pesticides exhibit subtle effects
on the environment. The Agency is required by FIFRA to ascertain
whether a pesticide "...will perform its intended function with-
out unreasonable adverse effects on the environment..." [FIFRA
sec. 3(cH5)]. Th* effects may, indeed, be unreasonable and
unacceptable, even if considered subtle by some observers. The
purpose of- this and other subdivisions is to provide guidance in
the submission of data and other information. From this combina-
tion of information, an overall environmental risk assessment
concerning the exposure and effects of the pesticide can be made .
Included in this evaluation is a determination as to the possible
effects on endangered and threatened plant species.
The preamble to the November 3, 1980 proposed Subpart J guide-
lines (FR Vol. 45, page 729495 provided examples as to the possible
uses of the information. Also, Subdivision H, Labeling for Pesti-
cides and Devices j . provides the guidance concerning various 'types
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of label limitations , precautionary statements «.. ' . ^
relating to phytotoxicity. -. **..****«***•, or restrictions
8* S^ of Test Data
- , prmry "» «-«•
- ~
C« Test Substance. .
has concluded that
t-aring-ua. products ^
ropra^
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active ingredient in its product if the same data are reaui-ed to
a
support the registration of an end-use product that o
be produced from the registrant's manufacturing-use products!
dS«ST! ? *f ~!T Pr°dUCt " * P*sticide P»duct bearing label
directions for immediate end-use as a pesticide.) Section 158.50
also provides that such data must be suHnitted by an applicant for
registration of the end-use product, except that the producer of
the end-use product will generally not have to •ubait or cite data
££*" "?„"•!"' to fozffiulate **» «*-»•• product. This decision
reflects the Agency's- expectation that manufacturing-use product
registrants will .b* the major source of registration 'data* .and
that end-use product formulators will, in most cases, heed to
supply much less data. This decision :: is consistent with the pro-
visions of,. and Congressional intent behind, sec. 3{ c) (2) (D) , of '
FO.FRA, which .provides that: • ,
No applicant' for registration -of >• 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 -
• (i). 'submit or cite- data pertaining to the safety
of such purchased product? or
(ii> offer to pay reasonable compensation other-
wise required by [f 3(cM1)(BS< of FZFRA} for use of any
such data. - J
~1JIfPliC:it ** **C 3-(2HD) ** Congress' expectation that it
™f* ?* •4"9i«trmBt ot the =«nu£acturing-use product who would
S£ £ *i9ni*ie«nt aaounts of data pertaining to the safety of its
product. (See, e.g., Sen. Rep. No. 334, 95th Cong., 1st
pp . . 8—9 . } . •
^» " **** r^iuirsments were imposed solely on regis-
trants of -
-use products, sec. 3{c){2)(0) might be read to prevent
the Agency fro* obtaining data on the grounds that the data pertain
to the. safety of a purchased product. . ^^"in
Teatlin
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6
the active
of the applicant- .
""'
protection test-
The test substance shall be the end-use' product or a
representative end-use product from the same major
formulation category :f or that , general use pattern.
Examples- of major formulation • categories are , we^-
table 'powders, emulsifiable .concentrate*, and qranu-
lars;. ^ df the; manufacturing-use product is usSSy
formulated into end-use .products comprising* two or
^ ^30rJ0mal^ioa =^egories, a separate study
f£ \t^fl0rm9& \±th *
ror each category.) . •.
•hp^d->.aet«» tha* the sutaission of data using the
end-use product in question is recommended asl?
better describe any phytotoxicity associated with that
4* Technj-eal grade vs. fommi;.*-* oroduct r™™,.r,^
,.
the representative .end-use product to .be used in iier 3 -he
of the technic-al cheaical in .tiers 1 and 2 followfSe intent
and
°f tectoical Aerial at the Tier t
,._ ., , • -J, at a level that is not phyto-
to dissolve the material, in water or other. suitable carrier.
_Area ghytotoxicity Testing.
1* Phytotoxieitv .and efficacy teatir^.
a confusion between those phytotoxici'ty
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. 7 ,
Subpart J and those 'normally performed in
simul-
separate the sheets of phytotoxicity and efficacy. Product
performance testing and target, area phytotoxicity testing are
ordinarily and may continue to be conducted simultaneously.
..2*' Jf*iirer of target area phytotoxieitv. ; The Agency has
^f h t*Tt "" ^atoxicity ^ta does not need to be
:SlfCaUSe'^.fe9iStraatS *" ««««"y willing to accept
overall responsibility of the product respect to efficacy and
SEST^"^ SeC* 3"(C)(S»- °— ^ Sidelines Le
provided to the registrants for those instances where 'data may be
n.GGQ€scl«
. B ,3' ~ w««d-free control plots. The weed-free or otherwise -pest
free control plots of proposed §f 120-2(1)' and 121-1{C) E 1) Civ) wer
the proposed e
the maintenance of weed-free and : pest-free plots, -he
.conmentars stated that this is. very difficult, ^practical, 2nd at
times may be even detrimental to the crops. Therefore, the defini-
tion of -pest-free" has been changed to only reconnnend conLol of
are
P°r exaffl^1«' ^ «ntrol process of weeds
vfeh* la^l- As stated in sec.
f Fina> a pesticide may be applied "...employing a^y
f 5tP cmtl°'15 S0t ^ohiiited * the labeling..;- m the
proposed Sttbpart J guidelines [proposed § 163. 121-1 (c) (3 )] , all
,
ew ; prohibited by the label would have been eval-
uated with.-resp.ct to pesticide application and movement in the
counters have stated that testing all
"•«**• ,S2t Prohibited by the label is impractical and
. r
T* u i3-disco^a^ because of the celic.t^ " *
of that situation. Therefore, uSe of some methods
of application which ^are found on the label, need only,, bl tested?
If ^a -worst case- application method can be readily • determined
prior to testing, then testing, may' be limited to that case. Sup-
port for the use of -.that method should be famished to the -Agency.
con.at.rs
. synergism) and serial
cations tests were excessive [§ 12l-1(b)C5) ^and (6)]. -he
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ea °ce «-1 °^*nuary 1982 has
eliminated, in most cases, the r
ea as
emnated, in most cases, the requirement to «uhi.4<.
the treated trees*
The reporting .of general population shifts in pastures' and
.
7* Subaecment Planting (rotational frn^^ Comm.ni..^.
sion J [f .121-(c}
are used ",tq evaluate, the phytotoxic
^ontart?et ^e* Phvtotoxieity' Testing
regmirenen^ for' non
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o
or
The Ag«acy is retaining Subdivision J nontarget area phytotoxi-
£ , ^f, th°Se SitU"ions **« ««» information is leSed!
tlsf ^ f fideS,a "* °f' Standards «* reporting, formats
! ta Vfhen th*y •" «<3uest**' several examples
^ *?** f6^*6* •*•« <1> »«*«*• Posed to endangered
ened pxants listed -by the United States Departmen- of
Interior, Fish and 'Wildlife Service; (2)' initiation of a rLuttable
presumption against registration (RPAR) where a phytotoxicity
problem may exist; and (3) where a specific phytotoxicity problem
arises when general - open literature data are not available.
: The Agency will inf orm the registrant of the, chemical in ques-
tion, concerning the phytotoxicity problem and the specific data
required to address the problems
2* Terrestrial species selection. a the proposed Tiers 1 and
2, seed germination/ seedling emergence and vegetative vigor tests
(proposed ;§,§ -.163.122-1 and 163.123-1), ten specific kind, of plants
were to , be tasted. This made the -guidelines 'somewhat - inflexible and
did not readily .'-permit the use of auch screening test data already
generated by companies. The selection now states. that soybeans,
command a dicot :root crop are to be' tested, and that seven other
test species :are .to be a balance of monocots and dicots. Corn and
soybean were . retmmfed, due to their economic significance and the'
quantity of - .pesticide -research performed using these species. By
increasing tliis flexibility of species selection, tests that are
normally performed by the developer/registrant during screening and
initial field/testing may •• often be used. This change will result
in -a significant: cost reduction for this test.
, , 3* agaatic species selection, several ccaraenters noted that
inclusion of :five aquatic species at the Tier 1 and 2 level can lead
to expensive and unnecessary testing. They suggested that only one
.species, probably Selenastrum eapficAmuMa. be tested at the Tier
1 level. ' '
After careful consideration, the Agency decided that this
species selection was indeed unnecessary and that the. selection
could be based on use pattern. • Selenastrun will be tested for all
terrestrial or aquatic outdoor uses, if an outdoor aquatic use
pattern is anticipated, the other four ao^atic species would also
be. used.
The aquatic species selection was based on those species -hat
have been, extensively tested and for which the growth paramete-s
nave been strictly -determined and specific strains are read-'ly"
available. For .these reasons Lestna gibba G3 is chosen over' Lemna
3anor and Selenastrua capricorrmtua over Chlorella vulgaris . The
diatoms are used because they have been shown to be very sensitive
to water pollutants, toabaena floS-3Cuae is chosen as a reoresen-
tative of a group of plants that can fix atmospheric nitrog'en.
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The Coverall selection was made to obtain a broad representation
of aquatic plants and provide some insight into variations of "
rr "* Plant es r
- Tier 3 ( dicots , monocots , ferns, etc ..) addresses the fact lhat
.plants other than .alga* inhabit acetic areas, Again thislest is
to note the variation of .effects U.e., tolerance or .resistance)
•to- the pesticide. • ' . ' ., ; . - . <"lce/
4> P°f*gea or application' level,.. Many •eonmenters .to the
proposed Sufapart j guidelines stated that three tiaes the label rate
was an. unrealistic quantity to be assessed for aontarget area
phytotoxicity, This statement was based on information from actual
uses and .exposures. In response to, .these comments, the maximum
dosage or application level was set at the maximum label rate.
Again comments were received that this rate was excessive and that
the rate should be based on environmental exposure.
.. It was not the. intention of the Agency to 'perform these tests
after environmental exposures had been determined or modeled, if the
registrant, however, decides to perform these tier tests after deter-
.mmation of the .environmental exposure, then a rate equal to at least
three tome* the exposure as found in the adjacent nontarget area, may
be used. It aust.be remembered "that the adjacent nontarget area can
be the adjacent desirable plant of another species 0-1 meter or 100
meters distant., therefore, the use of this exposure level must be
supported with' appropriate data.
On the other hand the use of the maximum label or environment -
exposure rate does not preclude the voluntary testing and submission
of -phytotoxicity data where the tests were performed using higher
rates, it is noted that dosages 'used during manufacturing screening
tests would -have a greater ^tenden'cy to . exceed' this required dosage
or application level, and would thereby . increase the probability of
acceptance of -these screening ' tests . . :
Mutagenicity Testing.
Since proposing the' concept of a plant mutagenicity testing
•scheme .in Subpart J, many - registrants.and other researchers have
expressed concern-that these tests would not provide meaningful data,
AJ.SO, no incidence of plant autagenicity has been substantiated for
_target.area crops, or nontarget area •plants. '
Several commenters suggested that this set of tests undergo an
extensive series of evaluations before this type of testing be in-
cluded in any finalized ruling. Also, commenters and others pro-
vided references which question the-validity of using plant muta-
genicity studies to evaluate huaan mutagenicity.
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11
withdS thl h"e COfflMntS' the Agency-has decked to
withdraw the requirement for the plant mutagenicity studies
extensive testing can!,* performed to show the aorl
usefulness f or ^ this requirement.
G* -ier 3 •Fiel<* Studies.
Several commenters noted confusion in the requirements of and
tne differences between the Tier 3 aquatic and terrestrial field
studies and the Tier 4 geographical and seasonal field 'tests. This
confusion was -generated by the tier progression statements where one
processed from Tier 2 to either Tier 3 or 4, depending upon a
plex set of progression requirements. '
«. ^* conflision' ^1 field. studies were combined
at the Tier 3 level with respect to either terrestrial field or '
aquatic field testing. Geographical' or seasonal considerations
are Deluded in the Tier 3 tests. There is no longer any Tier 4
testing. ,
fixation Studies.
All testing,, of, microorganisms was removed from Subdivision j
except for testing of alga*. Therefore, testing. of the nitrogen
fixation potential as affected by pesticides was removed from -
^SV«SK!?'f*. Thl* study'wi11 *• considered for inclusion in pro-
rS6d f"1^-"- « ^«H»f «ith pesticide-microorganism effects!
Comments received will b, used in the development of these require-
ments when this subdivision is prepared. -eqmre-
!• Sorption Study.
^The requirement for a sorption study as proposed in Subpart 3
was based on a theory of possible mode of exposure of aquat
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12
J. Spray .Drift si^-i —.'
tlon series from SubdiJSLn ren°Ved ^ sec
J and
K«. ?ier Progression.
1 ^ 9eneral *^eed -that the- BC10 vmlu. for the Ti«r
the .testa must be repeated ' *rr™tal effect of 25 .percent, then
sits: "--s
Commenters also stated that
»., not
eneral agreement does, not exist among researchera
"
S^S=SS~=3
or EC95
2 levels, basina nia-*.- -i^-i ^ __
3n use patt
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13
- The TM-jfiTmi-m dose. level has been reduced to the maximum label
rate or to 3 times the maximum expected environmental exposure •
L< Statistical Analysis
Several coasmenters stated that for the results to be statis-
tically significant more1 replicates and/or 'a greater population
size would be required. A basic part of scientific analyses is to
have sufficiently. large populations in order that the results be
meaningful. The Agency is making the selection of population size
flexible as each study would require a different number of indivi-
duals. It should be noted that each species has a different seed
germination and survivability rate which has a. direct bearing on
the statistical significance of the results. The Agency encourages
the use of the largest possible populations for each of the tests -
in order to approach the 90 to 95% level of confidence .with a
significance level of less than 0.10. The following references
are provided concerning sample size selection. '
Casagrande, . J.T., Pike, M.C. , and Smith, P.G. 1978. An improved
approximate .formula for calculating sample sizes for comparing
two binomial distributions. Biometries 34s 483-486 «~
Fleiss, J.L. 1973. Statistical; Methods for ........ Rates, and Proper-: ••-.'
tiona . John -Wiley and Sons, Inc. New York.
Snedecor, G.W., and Cochran, W.G. 1967, Statistical Methods , . 6th Ed.
Iowa State Univ. Press* Ames, Iowa.
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14
SUBDIVISION J — HA2ASD EVALUATION: aONTABSST PLANTS
GUIDELINES
Series 12.0: GENERAL
§-"•1-20-1 ' Overview.
™
runoff, .soil • erosion, : spr.y . drift/ etc. ) .
(A) Toxi=tty to pl«« in the t«5«t .r.a (j ,21-1),
'
• '• -
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15
registration of each . end-use product with the prescribed use pattern
and each manufacturing-use product .used '-to make such an end-use
product. •:• - • ..._.'•
(2) The -test substance- paragraphs state the kind. of pesti-
cide material' that Bust be used 'in each test. The test substance
for studies in this subdivision may be- the- technical trade chemical,
or a representative end-use product. Generally, each of these
test substances is prepared by the basic manufacturer of a- pesticide
chemical. , .
(c) ires*"*g to meet requirements. -Since studies found in this
Subdivision would ordinarily be conducted by the basic manufacturer,
pesticide formulators would not often be : expected' to conduct such
tests themselves to develop data to support their individual prod-
ucts. (See 40 CFR § 158.50 concerning -the formulators'' exemption. ) .
They may do so if they wish, but they may -also merely rely on the
data, already developed by the basic, pesticide 'manufacturer.
(d) "^gg-et "6* phytotoxicity testing waiver of rfemii^m^^ .
(1) The Administrator has determined that efficacy test, data include
target area phytotoxieity testing data, and that data ^submittal for
such testing may be waived, by his. authority under FIFRA Sec.' 3(e)(5)'
for most kinds of pesticide products. (See 44 FR 27938-27940, F-iday'
May 11, -1979-. J Such products generally -include all pesticides whose
uses result in direct or indirect application to plants in the target
area such .as agricultural, lawn, and garden usa.
(2) ^^ though the" Administrator will ordinarily waive the
, requirement for submittal of target area phytotoxicity test data as
indicated in paragraph (b)(1) of this section, he reserves the
authority to require, such data on a case-by-case basis whenever the
Administrator deems that such data are necessary to evaluate the
acceptability of a product for registration, if it is determined
that data phytotoxicity for a .pesticide -are -necessary, the Agency
will promulgate the. specific target area phytotoxicity data require
ments by letter to a specific registrant or by general notice...
(3) Thus, the guidelines in this subdivision should be used
by registration applicants as phytotoxicity teat standards and
phytotoxicity data reporting requirements when target4 area phyto-
toxicity data are submitted to support registration applications.
'The guidelines may also be used to provide guidance on testing
to support the claims and directions for use on product labeling
for products for which target -area phytotoxicitv data submittal is
waived. " •
men1
{8) Sontar?«t **•»« ohytotoxieity testing. (1) Data require-
s. Data concerning the determination of outdoor pesticidal
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16
or
(2) Testing aeh*^ . Tests to the lower tiers n »«* ^
-
wfR part 158. Where data are stiimitted to fulfil *-K^
ments of one subdivision ero«« ^.J^ " rui£i11 the require-
a*d. by 'the reglsS^t !« ?£ ! «*«r«nce« to that data should be
y »• .egistrant if the .data ^e also required elsewhere.
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registration applicant is referred to Subdivision
120-2 Definitions
.
*n
' (•) The term "SDx" means that ^-ite— ml
dosage required to d.«rla.atmlly 5"^ pi
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13
50 percent
Ch) • Th* titan ' "nontara»t
or
Ci5 The tern "peat-free"
possible.
as free of pests
are
can
°
l
* action detrimental
and
°f
"
thaty *.
response My occur during germinaion oh di^ c
maturation of plants, and m*v^~^^' ^±*f.««**i»tion, and
sp,cl.,.
desirable and imdesirab!.
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19
12(?~3 i*sic teat• standards.
• -
pr«c«ae.,=« ln the p.rjom.,,ce of «at particular
Si*
s-i*-*"^ = - =£S
§ 62-2. ; imits, if any, certified in accordance
used ;l
their purlST?.? S!°ref Mder conditions that
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20
to possess
«* che^cal properties of the test
levels u*«5iVin the study, it should not produce
or toxic effects to' plants. -r,. P««««
(S) Where 'the test substance does not readily-
(i) An -analytically pure_.. grade 'of an active; ingredient;
(11) The technical grade of an active . ingredient;
(iii) An inert ingredient of a pesticide formulation,
!iV> A C0ntaminant or ^«rity of an active or inert ingre
of » active or
(vi) The pesticide, formulation;
sections- of this subdivision: 121, 122, 123, and 124
- (2) Healthy plants aast be used. '
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21
(S) The population size of m&ch replicate or treatment should
be large enough to assure meaningful results. Sample
(section series 122 and 123) should
2S or 50%
£ ield
-
system of random assignment of the test plants
group's is- required. "
-
ss: s •
in=lui?« 5?1tPMgt' (1) M1 *5^P^t used in • .conducting the
xncluding ec^ip^nt used to prepare and administer the tesl aS
*,• ^(2J Th" aPPUc*tion equipment used in testing produc-s' in sma
field plot ^studies should be designed ro simulate convenSnal *S
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22
small plot spray.« can b« identical
ccdaidcial «•«•••»«•—-» .
should produce results
t. on
cial
on
plot
au
-------
21'-*
f« ., r** ^"^i^isa «nd composition of any>ehicles
(e.g., diluents, suspending agents, . and -emulsifiers ) or o2er
used 'in th. testing of -th, 'substance.
of »*i- reporting requirement
to Sabdi^ision o (f 61-1, §
(S) Ontoeated eostrol ( cheek 1 olots. Detail.^ ^M<- 4*4 •
used as controls fo'
et T*"* tO ^^ ?«sti=id« °^ cneMcal
etc.) :and cultural practices, . .
"™™
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24
CD) Turbidity (visual), conductivity (if possible . „
dxssolved o*yg« (for submerged plants only);^d ' ***
--
. . „.
^-11™^-
term st«S " C°nditi°M -^ ^ «*d for lon
C°nditi°M -^ ^ «*d for long
for th* duration of fi9
-------
"* *** iatervml fr*1"- *PPlications should be
products are applied. is a tank
wi , •
with identification and formulation for each product.
(ill) Timing of applic^i^.. when toe test substance,
particularly a herbicide, plant regulator, desiccant,- or defoliant
is applied to any desirable nontarget plaats within or adjacent^o'
rsf: piant>s sta9e °f «rowth °r *™u*"Jt..f
should be described .in test reports. •• •
Aerial applications . In. addition to the detrimental
times o£ *PPli=*tion (or
each product °r
Observations. (1).. Observation* should b. reported to '
^^ions, either inhibitory or, stimulatory, between the
organisms, and the untreated controi teat organisms.
°tOXiC S*mpt0mS {c^°«»^' necrosis, and
(2) Observations should be reported in sufficient detail as to
Pt"ali011 °f th«-rM»lt-
, to be
, should include the. degree or extent
r.^JJi^"? dlltfla"ntal or «»'•»• effects to be considered and
reported during the observation period of terrestrial studies include
Ci5. Stand or plant population,- ;
di.« 1°V*r*11 T±9°r of th8 -Pl«« expressed as height, weight,
diameter, length, or other similar aspect of -growth,-
malfoi^il Fhy*0t°xicity or visii^ symptoms such as discoloration,
malformation, desiccation, or defoliation;
(iv) Lodging of plants;
Effect on root growth and structure;
to
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26
sr.S S5
.„,, (*1, AV*Mt two »«tliods of evaluation (such as quantitative
and qualitative determinations) should be used in the S^tton If
^stxcide effects on growth- reproduction, and yielTof J^ts L
greenhouse and controlled chamber experiments, ^hen direct measure-
ments cannot, be made, *uc* as in large field evaluations , a zero
to one hundred (0-100). or^zero to ten (0>10) rating scate
"
u
S •ST(',n"J'r0
-------
- .27%..
(2) Statistical analysis is also useful in evaluation of
interactions resulting from studies supporting tank mixtures or
serial application* [See 121-Kb) (5-) aad (6)]. '
(e) References.- Copies of references or literature used in
modifying the test protocol, performing the test» making and inter-
preting observations, and compiling and evaluating the results
should be submitted. Copies of unpublished literature should also
be included. Copies of the recommended literature referenced in
these- guidelines are not required. •
(f)' Special test requirements. In addition to'the data
required in this subdivision, data from other tests may be required
by the Agency for-making judgments regarding safety to nontarget
plants* Such data will be required where there are special prob-
lems, such as a proposed pattern of use, mode of phytotoxic action,
or a unique chemical property. Methods are -usually derived from
those already described or cited in other subdivisions of these
guidelines-. . •
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28
Series 121: TARGET AREA TESTING
§ 121"1 Target
area
phytotoxicity testing.
generally will be waived by 40 CFR Part 158 to 3 the
end"USe Pr°dUCt lBtMd«1 for outdoofS
or outdoor planting of treated material [
T^0'11' n°ted in § 12°-'^5' the Agency y re
data fro* studies provided for in this section
fiil. this section need not be fui-
fon^ol ?ideS WhiCh Provide long-term or total vegetation
control, e.g., clean yard chemicals, desiccants and defoliants.
also 12)H Experimental use permits. The registration applicant is
also reminded that an experimental use permit may be
° '
e
for information concerning experimental use permits
**
(1) Test substance. The test substance shall be the
?T,r * repr"«tativ« «*-»•• Product from theam
formulation category for that general use pattern. Examples of
maor formula
use pattern. Example
formulation categories are: wettable powders, emul.i-
concentrates, and granulars . (if the manuffcturing-u^
" ** "
— - c"~^™»*«^ &WQ O2T uiw^ c ui
- - - separate study must be performed with a
end-use product for each category.)
(2) Test species. Those desirable target area or pest host
Plant species as listed on the label (for example, the crop Pl^t or
ornamental) which will be within the target area ihould bTtes'd
:he plant cultivars to be tested should include representatives of
the cultivars that are most likely to be used.
(3) APPUcation3 levels. (i) The minimum, maximum (or -he
greatest allowable concentration), and 2 times the maximum libel
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.29
application level or rate should be tested. Levels greater than
effect (or no-effect} level should alse'b* determined.
The multiples of the, application rate to be tested are
.us quantities of the,formulation in the label-recommended
ty of carrier (such as water), to. be used per land or aquatic
Products .with labeling which allows or recom-
H - ' r rec
the addition, of separately-packaged adjuvants to the spray
tank ;should be supported with data indicating any detrimental
effects- (s«ch as -increased crop phytotoxieity) which may result
from their addition to the. pesticide, especially a herbicide,
Plant, regulator, :d*siccant, -or defoliant. If a range of adjuvant
rates is' recomended , the maximum rates within that range should
be evaluated in conjunction with the intended pesticide product.
(5) Tank mixtures. When tank mixtures are reccamended on
product labeling, a study may be required on' a case-by-case' bas^s
to demonstrate the extent of antagonism and .synergism with resoect
to detrimental effects on nontarget plants by, -.the products' of tank
mixtures. Antagonism and synergism are best evaluated in adjacent
plots, where possible interactions are sub jected.to* statistical •
analysis. See | 164-4 of Subdivision N for 'possible- combined test-
-- •
't C!,}; s«»-^-*l agglieatiQRg. Data requirements for serial appli-
catioa(s) at one or more pesticide(s) preceding or following '
T ** saae crof>
are identical to those described in, paragraph .( b) ( S ) of this
section for tank mixes with respect to phyto toxic ity, when such
serial applications are reccnmended on, the label. See § 164-4 of
Subdivision N for possible combined .testing, . . .
{73 Ii£«- Th« test should be performed -in .greenhouses or
wherever the product is intended to be used.
' . (8) Protocol. The protocols, methods, or practices should be
S^LTP f^r ^ ^icipated registered use of the pesticide
product. Specific points of information that -should be addressed
concerning use patterns, application methodology,- cultural prac-
^T*!; r*8P°nSM' ^^ subsequent planring are -found in paragraph
(c) of this section. :
. ^*}~ JSF3^^?' In addition to the information required by
J_ 120-4, the test report should include the following information
7ltVKr!fP*Cl: to Ph^otoxicity to the plants within the target area
(with the exception of weeds). This information should include
the method of application, cultural practices, plant responses ,
subsequent plantings, and use patterns that may be involved.
J
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30
(1) S«aeralinformation. (i) Timing of.
f any pesticide-, their stage of growth or
at application should be described in the test report!
„. (ii) •M*t^relogic«l conditions. Where .meteorological condi-
tions cause detrimental effects on plants which in tarn allow the
pesticide to further adversely affect the plants, ' the specific
£S!±i'" *Jf J! t such as soil moisture content and
temperature, which. are directly affected by meteorological con-
ditionp, should also be reported. Soil moisture may be observed
azid^expr eased .in terms of • dry and cracked, waterlogged, or other
similar conditions. Organic matter content of the soil should
also.be reported.-' ' •
4J. fiii3 ggray Dilutions, .in foliar applications, when a pesti-
cide is applied as a diluted spray and the quantity is dependent
upon the number of trees per area or ^density of 'vegetation, the
total spray .volume per .unit area, and the concentration of. the
applied pesticide should be reported. '.' :
gntreated controls f cheeks). ,la phytotoxicity evalua-
tions, all treated plots, plants, and/ or ' commodities should .be
compared directly to untreated control plots, plants, or commod-
ities. All quality and/or yield evaluations of pesticide-treated
plants or ceamodities should be compared to control, plants or
commodities receiving the same pesticides (e.g., herbicides,
insecticides, 'fungicides) except the one being evaluated. Detailed
descriptions of plots and' plants used as control treatments for
comparisons of detrimental side effects should be included for
each test. Since such control 'plots are established to evaluate
any direct detrimental effects of the pesticide on the crop or
commodity rather than to evaluate efficacy, any detrimental
ef .ects on the crop or commodity resulting from pests should be
controlled, in other words, the control plots should be both
untreated by the pesticide in question and as pest-free as reason-
ably possible. If,, in addition to the untreated control plots
plants, and/or commodities, a 'registered product is applied (as a
standard) for ccaparison' of detrimental' effects, data should
indicate the -standard product ' a ' name', active ingredient, dosage
rate, and phytotoxicity results.' Where infestations of weeds
occur in check (or test) plot«,.th« degree of infestation and
species of weed(s) should be reported.
(2) Ose patterns. When the following use ' patterns are found
on the label, the corresponding information as detailed below should
oe reported. ' •
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31
in field • crops* Effects of pesticides on desirable
area .plants should be evaluated and reported* The extent
and duration of the effect should be expressed in terms of stand
and..vigor,_recovery, yields, and degree of .phytotoxicity.
;.lii) Use on pastures and rangelands. Effects of pesticides
on desirable target area plants should be evaluated and reported.
Severity and duration of 'adverse effects on desirable plant species,
expressed in terms of stand and vigor reductions,'recovery, and
changes in yields, should be reported* Data should be submitted
addressing reseeding intervals which minimize adverse .effects on
rcseeded plants, and animal grazing reeemaendatid'na. which allow
recovery of desired plant species. If the applied pesticide kills
all vegetation In the treated area for. an extended period'.of time
resulting in bare spots, 'the registrant should record the duration
of this effect, estimated /soil 'loss by erosion and any changes in
vegetation cover (desirable or undesirable)*
(iii)'Pse on and around fruit.and nut trees. Applications of
pesticides on and around fruit and nut trees require evaluation and
reporting,of detrimental effects-on foliage, and changes in, growth
compared to preapplication measurements and simultaneous controls.
Pesticide applications to bearing fruit and nut tree areas also
require evaluation and reporting of detrimental effects on yields
and commodity .(produce) quality for the year of and the year after
application. Supporting data should address, for all trees, the age
of the trees, the transplant-to-application interval, and the maxi-
mum allowable extent of contact between the' pesticide (with par-
ticular reference to herbicide'.spray drift) and trees. For ground
sprays, unless the .pesticide is broadcast over the entire orchard
floor, data should indicate the application technique .(band, spot,
shielded, or directed spray application) and the size of the
treated ground area .around the tree trunk* Assessment of root
sucker - treatments should be made where applicable. For foliar
sprays, the data should include the volume of finished spray applied
per unit of land area; concentration of product in the spray solu-
tion, and the extent of foliage,coverage (such as volume of finished
spray per tree or application to the point of runoff).
(iv). , Ose- on.-:lawna, and turf. Evaluation of'effects of pesti-
cides on representative species - or,cultivarS of desirable lawn and
turf plants should include, such factors as color, density, percent
.cover, growth rate, •,rooting, and tillering. If use on bentgrass
is intended, this highly susceptible species should be evaluated.
Data should address- use--on newly-seeded lawns by demonstrating
safety to representative species and cultivars of desirable lawn
plants to b« named on the label-as kinds on which the, -product is
safe to use, with seeding-to-application intervals-(if appropriate).
Data should also,.address-.-use of an appropriate application-to-
reseeding interval for each of these desirable lawn plants that
may be reseeded. Interactions between herbicide application and
J
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32
lawn cultural practices•(such as raking, mowing, mowing height/
watering, and fertilizing) should be evaluated for possible
adverse effects on'desirable lawn species. In situations where
fertilizer and a pesticide .are applied serially and both types
of products may contact -the emerged crop foliage (such as in turf
or lawns), the interval between application of the pesticide and
the fertilizer should be reported, as well as any resultant phyto-
toxic effect, stunting, or discoloration, and recovery time for
the injured desirable species.
(v) Use around ornamentals. Phytotoxicity data"in support of
use on or around an ornamental should include an evaluation of the
sensitivity of representative cultivars of that species. Since it
has been documented that cultivars and varieties of the same species
vary in their susceptibility,to 'injury, the limited nature of test-
ing should be addressed in product labeling. Test data should iden-
tify the method of application as to"'1 directed spray and/or topical
applications. Growth stage of the ornamentals and the transplant-
-to-application interval (when applicable) should be indicated
in ~.ne test data. Information should be submitted on specialized
nur -«ry cultural practices employed in tests, -such as use of
arr-ficial.soils, mulches,,containerized stock, and other pesti-
cides. ; •, '
(vi) Use in forest management.1 The effects of the pesticide
on desirable plant species commonly present in forest management,
in addition to the desirable forest trees, should be indicated in
the'report with any detrimental or adverse effects "that the pesti-
cide, may cause. Special attention should be given to pesticidal
effects on noncompetitive ground cover species that-aid in the
land management practices such as erosion control. Appropriate
testing and assessment techniques adapted to the size of the plot
should be used to determine the effect of pesticides on all plants.
(A' recommended reference • is: Phillips, E.A. 1959. Method's of
Vegetation Study. Holt,.Rhinehart, and Winston,' Inc.: 'New York,
N.Y. 107 pp.) , - ' : - ; ;
(35 Application methodology. All methods o'f'pesticide appli-
cation specified on the -label should be evaluated and -reported.
Specific detail as to descriptions of equipment design, adjustment,.
and operation should be :provided in test reports involving aerial
applications 'and applications-using 'conventional -farm equipment
_(such as tillage or planting equipment), irrigation systems,
mechanical incorporatiori, directed sprays, mist "blower (air
blast, air carrier), subsurface1-placement, or band rather than
broadcast distribution. . .... ; ,':•:,, :
(i) Aerial application. Guidance and the data requirements
for 'testing aerial applications,,will be provided in a subdivision on
spray drift exposure assessment.
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J
33
Irrigation system
fi
field should be examined and the
oh
of hax^estafale ccanodity) as an indication of pestic
Data from such plots should ;b. reported for eaS S
- f '
**** Spriakler '*•«•*»» overlap. On a e«nt.r-
w t0 ^ 38Verml "Pie" «««10B.-for treatment
subplots m one half with the second half -as the control. The
concentration' of. -active ingredient at .several .no«le8 alon/?he
lateral should also be determined and reported-. ,
CC) For .surface- irrigation systems such 'as flood, furrow dr
and surge, the following data should be submitted. ConcenSatlo^
!
;
applications. Guidance and the data
blast «- *^ carriers^
, ers w
provided in , wbdivUlon on spray drift exposure assessment
t«m, volume of liquid' or gas applied p unit aref^or gen«
broadcast applications - or linear row dfst«ce for band aid row
-------
34 • •
.^iSLT^IbHs'S^^*^^
'^'^rssisx'tsr,"^to '*y °- *«*«•
h.li=opt=r, or fi,.d-wL, ScrS° ' P P1°9
-------
35
«op
prior to or ju,e .rt.r a p.sticia. application ««y, by
d.sira.,1. ,!„„ or by d^Lia, 5ro«h
practices should be reported la teat results.
_ (5) Tafget area PlaBt '••"""*;.- The ..detrimental effects on
crops, coonodaties .(produce)., -or any ..otker^sirable ^plmnt s-pecies
TLC?TT f Within ^ target.ar.ea.sho^^.^eval.oat.d a^d "
Th. following are some of .the characteristics - that should be
{1) Stand, crop stand counts, reported as percentage of
SSrif irStr°1 "^ St*ndS' •he«id"b« suinitted to support pesti-
cides.- applied prior to crop -emergence. - -.*'*- ±«»*.A
^ij , -Zi22E'"" C^OP vigor (or stunt) .ratings or measurements '
ta cot tf J' T ?ht' dlafluster' « l«»9th) in treated areas should
SveS*^6! ? Plants in check plots in which commercially acceptable
levels of pest control are maintained. Vigor ratings should be
I?ir2Dort± ?ff °f mmXimm •ta»tl»9- If -Bunting is observed,
it is important that subseguent evaluations b« made to document the
oagree- of recovery. . - . •
„„„ Uii) Pi"ttn! ^P^.' A ran?e of P^ting depths' within the
range recommended for the crop should be included ii preliminary
"
Preemergence (to crop) applications. Data
from these 'trials should reflect any effecls of varying
planting depths on .the' incidence of crop injury that might be
r^T CaaM«i*1 »" condition,!- L subset trials,
T? T ^r^f1"*^ St '•«— ««« ***** settings should
if in preliminarv studies the planting depth 1.' found to
!1 ^*iafal«' ="p emergence data should be taken 'from all
effect of pesticides on lodging of target
Arcane
ippr°Ximate d«^«* °f "9l« of lodging in treated
should be. compared to that in weed-free check plots.
(v)
ghvtotoxieity. Evaluations of visible, symptoms of pesti-
=ide mw (such as discoloration, ^formations, Ssiccation
defoliation, or death) to crop plants should be at least visually
assessed and reported. These symptoms should be compared to results
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36
in check plants untreated with the a«st±=H A. <„
a^aas: sisS
£•=2 ,— - ~
report^3 Hef? L*8 •'P^ici— onyi-ld. should be
b8e<^eat Planting. The effects of «.«*-)<-<<»
-------
37
(ii) Rotational crops (including cover crops). If detrimental
effects are observed, results of studies evaluating severity and-
duration of effects on the injured rotational crops should be sub-
mitted. To determine the duration of phytotoxic effects,-susceptible
rotational crops should be planted at varying tine intervals after
pesticide application. Such studies nay be combined, with field
studies designed to evaluate soil residues. [See § 165-2 of Sub-
division N.]
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38
Series 122: TISR 1 OP NONTAHGET ASEA TESTING
of
Dieptyledon«m.i • six
-P.C1..
,# =,,.
3r=,»p, should
•
that .,u.
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39
target area is significantly less than the maximum label
rate .
°f ««l«l-t** the dose level « the' seed
'- - ° artW ia^edient P« «« should be
considered to be equal to 3 ppmw in the solution which is applied
to seeds. (Notet a 1 lb. ai/acre application to a 3, inch soTl '
depth would equal 7.5 ppnrw in the soil solution.)
'*?* ° plaa^- At 1«*rt 3 replicates, each with
st = each *ith at least 10 seeds, should
be tested per dose, letrel for the seed germination study. Larger
populations and »ore xeplicates may be needed :to increase the
statistical -significance of the test,
.H~ T I5 il ^' Th* seed- -?ermin«ti«/s*«'iling emergence studies
should be conducted under controlled conditions in growth chambers
or greenhouses. The vegetative vigor test may be performed in a
growth ehanber, greenhouse, or in small field plots. *
<«> P««ti«i. Ci) Seed gemination, if performed usina petri
plates or seed germination paper, should be assessed after 5 days.
Seedling emergence should be observed weekly/ or more frequently,
for at least two ^weeks after germination.
Ui) The effect of vegetative vigor should be .observed weekly,
-fe-; ' anora* symptoms
, the observations should be continued until the plant dies
or fully recovers. ,
C7> Protocols. -Hie protocols for these tests outlining the
acceptable environmental conditions , procedures , and some pertinent
references. ..are found in § 122-30 (a) through (c). '
_ (e! I Reporting, m addition to the information required in -
I 120-4 (b), the test report should include the following iziforma-
tion. - •
(1) The number of seeds tested and the number germinated or
emerged per dosage level for each replicate;
ment t** * *** ^he growth and develoo
ment of the seeds and emergent plants, indicating any abnormalize.
and expressions of phyto toxic ity; and '
(3) Tabulation of the results indicating the percentage
Sl f°r MCh Sp*Clla as conf«red to untreated control
-------
also
40
and height or other growth parameters may
. .' {d> Tieg orogrwioB. (1) 2f the results of the seed
germination/seedling emergence test(s) have indicated an' adverse
effect greater than 25 percent: on one or more plant species ,. then
seed germination or seedling emergence tests at the • Tier 2 level 'are
reqmred, { see }- 123-1}.,',, • •. '.' • • ':'' . V
,. i. A lf-thm «salts of the vegetative vigor test(s) have indi
cated an adverse effect greater than 25 percent on one or more
plant species,: then vegetative vigor tests at the Tier 2 level are
required (see § 123-1). '
_ _ (3) If .less .than 'a 25 percent detrimental effect or response
is noted for .either seed -germination/seedling' emergence or vegeta-
tive vigor tests, no additional testing of the respective tests
at higher 'tiers is ordinarily required. The Agency, after review
of tne data, may require certain additional tests to determine a
more definite nondiscernible effect level.
$ 122""2 Srovth and reproduction..ef aquatic 'giants (Tier 1) .
_ U> ' ?*** required.. (1) Data on the toxic .effects of a pesti
c^» on growth and. reproduction of aquatic plants are required by
40 CFR Part 15:8 •aa_«-fiOfcbZrcM« . basis to .support the registration
of each end-use product intended for outdoor pesticide application,
and. each manufacturing-use product which legally could be used to'
make such end-use products. I See § 120-1 (e).]
(25 Studies of this section need not be conducted for pesti-
- cides applied by systems where the chemicals are not readily
released into 'the environment. Examples of these systems are-
tree injection, subsurface soil' applications, recapture systems,
and wick applications.. ' *''*'•
(3) Portions of this Tier 1 test may be combined with the
respective parts of the Tier 2 test {§ 123-2) and performed as one
•
(4) See § 120-1 (e) concerning substitution of testing and
data submission requirements. ' . - . -
(b) 7est standards. • In addition to the general test standa-ds
set forth in §,120-3, the following standards for the studies of the
growth and reproduction , of aquatic plants apply: • '
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41
ILifT '! ^-' The tecimical 5r^« of the
f * .*•«*• Where « technical grade does not exis
the manufacturing-use product: or an end-use product with the h±«h.,t-
percentage of the active ingredient shall be usedf *^h *h* hi?hest
(2) Species. (j)" 'Selenastrum capt-i^r..,^ /.,' «T..^
a) should 'be tested regardless of the intended outdoor Le
(11) If the intended use pattern' is -for. outdoor aquatic pest
...Lemna gibba .(-duckweed) ; .....
Skeletonena eostatum f marina '
A freshwater diatom (unspecified species),, and
Anabaena fles-aonae (blue-green alga).
t6^J35, ^ff1^8^011 levelS- a« ^
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42
Protocols. •' The protocols for these tests
C0nditi°-
found in §
addition to
r Procedures aso
references are found in § 122-3Q(d) through (h>?
by
ml J!L MS£* - Growth should b* expressed as the cell count per
ml, biomass p«r volume, or degree of growth a« determined by '
spectrophotometric- means; and '
(3) Tabulation of the results indicating the percentage effect
level versus tine as compared to the control. Percentage effect
(d} ?ier Progression. (1) If a detrimental' effect 'or
controls, Besting at Tier 2_is required. See | 123-2.
(2> If less than a SO percent detrimental effect or
is noted no additional testing at higher .rates '
t ' ra
tests to determine a more definite nondiscernible effect
^ r22~30 Aceeptalale methods, and reference.
= r.-rr ss.
Chemical- 0* — S- -7 be 'surface-
seeds
onti tCSt te»P*rature should approximate the
optimum temperature for the species and variety used.
after^five days or more frequently.
germination is reported as th* number of germinated seedT '
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43
references.
B°*awit*' M' 1966* * rapid. bioassay for PEBC and its
TOlatil4»«*°* «* *i*orption studies . jellies!
' The use^of three
bi°*»say, on forty-two herbicides. weed Sas. ii
(iii) TrualOTa, B., (ed). 197?. Research Methods in
mir emerqenc<- (1^ Protocol. (ij seeds may be'
ft fTS Antacid-wash^ .sand or -a standardi^d soil.
least 10 seeds per pot should be used. .The seeds -may be surfae
'
treated with a known quantity of ~th* chemical. The test
should approximate those optimal condition, for thl sp^c
^arxeties considered. The seeds should b« incubated for at- least
Udays. The seed, are observed after 10 and 14 day," and
««rgenc. is recorded as the number of emerged seedling,.
Of ^* ^*;tMt M2T b« extended by 14 days to assess the effect
of •"^ «PPH«d-p««ticid«« on vegeta-tive vigor. ' '
^2' Reeomaended reference. • '
Southl~r«rr!'cB;' {€d)* 19?7' Research Method, in weed Science.
Southern Weed Science .Society. Auburn Printing Inc., Auburn, AL 221
, ,< (C) H«g«tati*e vigor -foliar gBMy. (1) protocol. Ci) ^
roiiar spray,can b*.applied, by .any acceptable method using labora-"
°=ry-, greenhouse-, or-field-grown plant,. . The plant should be 1 *n
4 weeks post-eaarg^t in order to gain young folfag^? ?Ss 2
^^ .M method, of, foliar applications may be found irfthe
»xieT* (^r^iT^*1 effeCtS are t0 be reP°"ed as severity
snt /
of
tne seedling emergence study. '
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44
(2} Mconmended reference. Truelove, B., (ed)'. 1977
r=h Methods in weed Science. Southern Weed Science Soci
Auburn Printing Inc.,. Auburn, AL 221 pp. «=-
(d) Lemna qibba; Growth conditions. (1) Species and
|ibba G3. Source, Dr. Charles Cleland, Smithsonian Radia-
Biology ;Labosatory,, Rocterill,,. MD ^20852 (limited supplier)
(2) Protocol. The following are acceptable conditions for the
growth and maintenance of Lenma ' gibba G3.-'*
'' (i> Environmental conditions.
Light Intensity s 5 klox (approx. 100 uE aT2s-1}
Light Quality; warm white fluorescent
Photoperiod? continuous light •• "
Theraoperiodj continuous 25 + 2"C
Eii) ^: '-'•sre conditions. . '
Liquid culture • •
Nutrieatss M type -Hoagland's medium without EDTA or
sucrose (Hillman, 1961 a & b)
pH S.O * 0.1 after autoc laving
(114.) Procedures. The vessel size- to-mediun quantity ratio
should be S to 2. Maintain the Leana stock under axenic conditions
!!«!.*?*** %f7 ^ '^rfortt«1'un<5« aon-axenic conditions as long as hbn-
organic media are used. Sucrose £10 g/1) and EDTA (9 mg/1) may be::t;
added if flowering is desired.
references.
(i) Davis, J.A. 1981. Comparison of static-replacement and
flow-through bioassays using duckweed, Lemna gibba-G3. o.S.
Snvironmental Protection Agency. Washington DC (SPA 560/6-81-003).
(ii) Hillaaa, W.S. I961a. Experimental control of ' flowering
in
III. A relationship between medium composition and the
opposite photoperiodic responses of L. oerpusyilla . 6746 and L.
gibba G3. Amer. J.-Bot. 4a;4l3-4i9. . • • —
(iii) Hillaaa, W.S. 1961b, The Lemnaeeae, or duckweeds. •
Bot. Rev. 27:221-287. '. - --
capricornutua: Growth conditions. (1) Soec' es
Selenastrua eapricornutua Printz. Source: EPA Corvailis Laboratory
Corvallis, OR 97330 . • y'
<2\ Protocol . The following are acceptable culture conditions
-or the growth and maintenance of Selenaatrua caoricomuttia.
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45
(i) Environmental conditions.. .
Light Intensity: 4 klux (approx. SO ut m-2t-tj
Light Quality: cool white -fluorescent
Photoperiodj continuous light
Thermoperiod: continuous 24 + 2"C
Culture conditions.
culture
Nutrients: U.S. EPA (1978) medium (EDTA shall not be
used in the experimentation medium.-)
pH 7.5 ___
O> Recommended references.
U) Environmental Protection Agency, National Sntrophica-
tion Research Program. 1971. Mfal Assay Procedure: Bottle Test
BT* Nit±0nal •"'i'oa^ntal Research Center, Corvallis, OR
(±1) Millar, wsfc, j.c. Gr««n
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46
£3) Recommended references. .
.U5 U.S. Environmental Protection Agency. 1973. Bioassav
procedures for the ocean disposal permit program, g.s. SPA Labora-
tory, Gulf Breeze, FL 32561 ( SPA-60 0/9-78-010 ).
(ii) Walsh, G.E., and S.V. Alexander. 1980. A marine algal
bioassay methods Results with pesticides and industrial wastes.
Water, Mr, Soil Pollut. 13:45-55. .
(9) A Fresnvater Diatom; Growth conditions. (1) Soeci-s. (To
oe selected.} . . ' • "
(2) Protocol. The following are acceptable culture conditions
for the growth and maintenance of Navicula seminulum or other selected
.freshwater diatom. • ' ' ~" - "
(i> Environmental conditions .
Light intensity: 4.3 klux (approx. 85 uE m-2s-1)
Light quality: cool white fluorescent
Photoperiods continuous light
Thermoperiod : continuous 24 + 2*C. ' ' '
Culture conditions.- • '
caltar*
Ntitrientss U.S. EPA ,{ 1971 ) medium
pH 7.5 • ' ' '
(3-) Recommended reference. ' •
Environmental Protection Agency, -national Eu^rophication
Research Program. 1971. Algal Assay Procedure: Bottle Test
(AA»:BT). National Environmental Research Center, Corvallis,
OR -97330 • ' .
(h^ Anaba«na flos-aquae; Growth conditions. (1) Species.-
Anabaena flos- aquae (Lyngb.) DeBrebiason. Source: EPA Corvallis
Laboratory, Corvallis, OR - 97330 '
Protocol. The following are .acceptable culture' conditions
.for the growth and maintenance of Anabaena flos-auuae.
conditions.
Light intensity: 2 klux (approx. 40 uE m~2s~1)
Light tjuality: cool white fluorescent
Photoperiod: continuous lighr
Theraoperiod: continuous 24 * 2 "C
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47,
(ii) Culture conditions.
Liquid culture
Nutrients: U.S. EPA (1978) medium (EDTA should not
be used in the experimentation medium.)
, pH 7.5 (not to b« exceed 8.5) " .
(3)- Recomn.end.ed references.
(i) Carr, N.G., and B.A. Whitton, eds. 1973. The Biology
of Bluegreen Algae. University of California Press, Berkeley.
676 pp. . ' - ' - •
(ii) Environmental Protection Agency, National Eutrophication
Research Program. 1971. Algal Assay Procedure: Bottle Test.
(AAPsBTJ. National Environmental Research Center, Corvallis, OR
97330
(iii) Miller, W.E., J.C. Greene, and T. Shiroyama. ^1978. .The-
Selenastrum eapricornuturn Printz algal assay bottle test. D.S.
Environmental Protection Agency, Corvallis, OR 97330 (EPA 600/9-78-
018). ' • .-. . -
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48
Series 123s TIER 2 NONTARGST AREA TESTING
* 123~1 Seed germination/seedling emergence -and veget
-- ;
r 2
required. (1) Additional data on the phytotoxic
effects of a pesticide on seed germination/ seedling emergence or
vegetative vigor, respectively/ are required by 40 CFR Part 158 on
a case-by-case basis when a 25 percent^phytowxic effect S one
or more .plant species is noted as -a result of the respective Tier
1 tests. These data are required. to " support the registration of
each end-use product intended 'for outdoor application'..
.(2) Portions of this Tier 2 test may be combined with the'
respective -parts of the Tier 1 test (§ 122-1) ;and performed as one
&6S-t» • •'
(3) See § 120-1 (e) concerning substitution of testing and data
submission requirements. • . "«&«
(*l 7es* standards. In addition to the general test standards
set forth in § 120-3, the test standards for this section shall, be
the same as those contained . in the Tier 1 studies If 122-Kb)] with
the following modifications: -
(]} P°«*?*«- Th« following dosages should' be tested: (i) At
-least 5 dosages should b« tested?
(ii) 2-he dosages should include a subtoxic «EC50) and a non-
toxic concentration;
(iii) The highest dosages should be less than the -1-fold dosage
tested in $ 122-1
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J
49
is greater than the BC25 for one or .more
123"2 Grovtn a**3 reproduction of aquatic plants
__ i,,aV0'*9"* Sh0llld ^ °f 9eoffietei= Progression of no more
ff ?*- '" ««»Pl«» the test concentration series may be- 0.1
.2, 0.4,- 0.8, and 1.6 kg/ha/15 cm (a 2-fold progression).
r/, (2) PIant «o«gi". At least those plant species of Tier 1
22"1211 %*iCh «hiiite
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51
Series 124* TZ2R .3 NOtnSURGET AREA TESTING ' " •
T
5 124~1 Terrestrial field tasting (tier 31. •
_u (*} • WMa g«T"*g«fl» CD Data on the phytotoxic effects of
•»!-«• product on Mad germination, vegetative vigor, and
reproduction potential under field use conditions ara required by
~S SJhP*!L15a °n ! c*»«-by-c*~ *•«*• to support the registration
of each end-use product intended for outdoor -application, the
a«i«u=. racomesded application quantity or anticipatad environ- -
mantal axposure ia to ba actual to or graatar than tha BC2S for ona
or aora tarrastrial plant species as found in tha Timr 2 ta«ts
(| 123—1) »
„,, i2i Th* idata r«Ittir«*=*s °« thi. section need- not ba ful-
filled for pesticides applied by systems where tha chamicals are
not readily released into the ararironnent. Exaaples of these
systems are: tree injection, subaurface soil applications, recap-
ture systems, and wick applications.' *
(3) See } 120-1 (e) conceraing substitution of testing and data
requirement submission. *
set fo^h !!*! Sf*;' fc Mliitloa to th. general test standard,
sat forth in § l2§-3, tha test standards for this section shall be
S! JT' S th!^,!?ntalJl*d ^ f 122-1(b> «« ^» subdivision, with
tha followiaf modifications!
„ (1) ?Mt substance. The test substance shall be the end-use
product or a representative end-use product from the same major
formulation category for that general use pattern. Sxamples of
major formulat^m categories aret wettable powders, emulsifiable
concentrates,, and granular*. (If the manufacturing-use product is
Mjo^LlSiSr1 iat° *ad"U" V*0*"** <=°»pri*inrtw6 oTaS
a typical end-use product for each category.) ** °m
- „. C2) *PP^.c«tion- levels, fhe dosages tested should be the same
as those employed in the tier 2 test [f 123- Kb) ( 1 )] .
_ t3) SiSSiM*- (i> Hapresentatives of the following plant
UiiTbalowf * fe**t;6d' *Ui'J*Ct *° th* llmit*tion« of paragraph
(A> Oicotvledonae (dicots) , representatives of three families?
(B) Monocotvledonae fmot,^.^.^ r representatives of three
families}
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52
(C)
two famili ' naves of.
^D) 33T°phyt« Cesses) or HepatODhyta_ (liverworts), one
sentative (for wetland use., pattern* only )~and
(2) gymnospermae (conifers), one representative. '
Cii) Plant species . used for testing Tiers 1 and 2 can be used
to satisfy the aonocet or -dicot. test plant requirements of this'
" " - • "••
to t not likely to be exposed
to the pesticide under normal conditions' of use, 'testing of such
groups is not required. Justification for elimination of a test
species or group should be included in the test report.
,., , u - sP«ci« ^y b« required if the general
selectivity of the pesticide cannot be readily identified.
J4) ^st conditions, "giants are to be grown under field-use
conditions similar to those of the natural habitat of the plants in
use.
^5} Duration. The test duration should be of sufficient length
-to assess multiple applications directed by the label. Obser^tioS
should continue^ for at least two weeks after 'the last application and
for * maximum of four week* to note any recovery or death. '
(6) Sea**?" °f application. The test substance is- to be applied
over a period of time or season according to. the proposed label
instructions . . . - . • .
(7^. T««* locations. The pesticide. -should "be tested in .those
geographic locations where it is expected, to be used, as based on
proposed label use sites. Where important species diversity and
physiographic differences occur within a region of intended' applica-
tion, regional testing may.be inadequate, and testing at a more
specific region or biome level may be required. United States
regional areas of potential testing' include:
Northeastern temperate deciduous; '-' '
Southeastern temperate • deciduous;
Northern grassland. {prairie );
- Southern grassland ( prairie},- '
Northwestern (and Alaskan) conifer forest and high desert;
Southwestern chaparral Mediterranean and low desert; and
Hawaiian and Caribbean tropical regions.
Cc). Reporting,. In addition to .the information required in
§§ 120-4 and 122-1(c} of this subdivision, the test report should
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53
include the test condition* employed (including the soil and
environmental conditions) and the determination of 'the 50 percent
detrimental effect level. • ' F«^c«Jt
5 124~2 ^^uatic field testing (Tier 3).
(a) ***** required. {1) Data on the phytotoxie «ff«cts of the
product on growth and reproduction of an expanded number of aquatic
plants are required by 40 CFR Part 158 on 'a case-by-case basis to
support the, registration of each end-use product intended for outdoor
pesticide application, whens
(i) The anticipated environmental exposure is greater than the
SCSO for any one aquatic plant species tested in Tier 2 tests {§ 123-
2); and '-....
Cii) The pesticide is expected to be applied' to a fresh water,
estuarine, or. marine aquatic system by -either direct application ox-
direct discharge of treated water (except swisHing pools) , or the
pesticide is to be used within a forest system. [See f 160-3(c),
(d), and (e) of Subdivision R for descript±ojss-of these aquatic uses.l
Pesticides- with only terrestrial uses need not be tested.
(2) See | 120- 1(e) concerning substitution of testing and data
requirements submission.
(b> ***** «*«pq*rds. In addition to the general test standards
set forth in $ 120-3 of this subdivision, the test standards for this
section shall be the same as those in f 122-2(b), with the following
modifications!
C1) T**t «ofeyt*nce. The test substance shall -be the end-use
product or a ' representative . end-use . product from the same major
formulation category for that -general use pattern. Examples of
major formulation categories ,arei wettable powders, emulsifiable '
concentrates, aad graamlars* (If the manufacturing-use product is
usually formulated into end-use products comprising two or more major
formulation categories, a separate study must be performed " with a
typical end-use product for each category.) . -
Application levels. The dosages tested should be the sane
as those specified in the Tier -2 aquatic test standards [5 123-
Kb) nn. - 5
(3) SP«cie_s. (i) Aquatic plant representatives of the
following plant groups are to be tested-?
Pieotyledonae (dicots) , one representative?
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54
families, -n°COtVled0na* <*«»«*•>' representatives of ' three
of
Division, * -°E>, a representative of each
Bryophyta (aosses) or
(ii) Plant species used for testinc- Tiers
the
(iii) Additional plant species may be recniired if
selectivity of the pesticide cannot
°r
by naturU or
..y. b. n.c.,.ary to .i»ul»t. the reduced l-
c«txoll.d
••
(Y). .The. field studies should be
conducted using:
southds Ci ^ Methods in weed science, 2nd Ed.
Southern rfeed Science Socx«y, Auburn Printing inc., Auburn, AL; or
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55
(B) & protocol with prior approval of the Agency.
'Th*> teat duration should be of sufficient
length to assess multiple applications directed by the label*
Observations should continue for at least two weeks after the last
application and for a maximum of four weeks to note any recovery
or death. '
(6) Season of application . The test substance is to be ap-
plied over the period of time or season according to 'the proposed
label instructions.
{7} Test locations* The pesticide should be tested in those
geographic locations where it is expected to be used, as based on
proposed label use sites* Where important species diversity and
physiographic differences occur within a region of intended appli-
cation, regional testing may be inadequate, and testing at a more
specific, region or biooe level say be required. United States
regional areas- of potential testing include:
Northeastern temperate deciduous;
Southeastern temperate deciduous i
Northern grassland { prairie) j
Southern grassland (prairie);
Northwestern (and Alaskan) conifer forest and high, desert;
Southwestern chaparral Mediterranean and low desert} and
Hawaiian and Caribbean tropical regions.
(c) Reporting. In addition to the information required by
§f 120-4 and 122-2(c) of this subdivision, the test report should
include the test conditions ( including soil , water, and environ-
mental conditions) and the determination of -the 50 percent detri-
mental .effect level.'
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P&CS
Pesticide Assessment Guideline, Subdivision J
Hazard ^valuations Nontarget Plants . ,
Robe
Hoist and ThomasC. Sllv*naer_
Office of Pesticide Programs
"J.S. 'Environmental Protection Agency.
Washington, 3«C« 20460
4. »»oort C»t«
October 27, 1 982
10.
JI. tswtnMsttC ar c/»m(O)
(G
12.
tna
Office of ?*sz;icide Programs
"J.3. SavirsnauBRtai ?roracrion Agency
'ro» a* atoart 4 »•«•«' .Co«»r»a
Guideline
iiincssn, O.C. 204SO
'U.
,
3-uid«lines Project Manager : Robert K. Hitch
Ao»t.-ict i
Suisdivision J, a Federal Insecticide, Fiangicide and, Rodenticide Acr
(FISTUV) .guideline, prescribes the phytotoxicity sttody protocols that the
U.S. Environmental Protection Agency recommends to support the registration
of manufacforing-oae- and formulated end-use products. This subdivision
establishes procedures for testing and data submission concerning the effects
of pesticides on nontarget plants. The studies outlined by this subdivision
will not be required for.every product but only for selected chemicals on a
case-by-case basis. A stannary of the rulemaking phytotoxicity test requirements
can "be found in 40 CPR -Part 158. The results, of the phytoxicity studies
together with environmental fate and efficacy are used to assess the potential'
hazard of pesticides on'terrestrial and aquatic nontarget plants.
Subdivision J constitutes one volume of a twelve-part FIFHA guideline
series published by the National Technical Information Service.
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