-------
- .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 Copies of unpublished literature should also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
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34 • •
.^iSLT^IbHs'S^^*^^
'^'^rssisx'tsr,"^to '*y °- *«*«•
h.li=opt=r, or fi,.d-wL, ScrS° ' P P1°9
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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 «.«*-)<-<<»
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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
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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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