United States
Environmental Protection
Prevention, Pesticides
and Toxic Substances
EPA 712-C-96-151
April 1996
&EPA Ecological Effects Test
OPPTS 850.4000
Plant Testing
"Public Draft"

This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 U.S.C. 136, etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP, or call 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and

OPPTS 850.4000 Background—nontarget plant testing.
(a)	Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the Federal Insecticide, Fungicide, and Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances Control
Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is OPP 120-1 Overview, 120-2 Definitions,
120-3 Basic Test Standards, and 120-4 General Evaluation and Reporting
Requirements (Pesticide Assessment Guidelines, Subdivision J—Hazard
Evaluation; Nontarget Plants) EPA report 540/09-82-020, 1982.
(b)	Introduction—(1) General. This guideline provides general in-
formation and overall guidance for OPPTS 850, Group D—Nontarget
Plants Test Guidelines. Series 850 deals with data submittal to support
registration of all outdoor use pesticides that come in contact with plants
and addresses testing for adverse pesticidal effects to nontarget plants, in-
cluding those which are within the pesticide application target area (such
as crop plants which are growing with weeds or are hosts for insects and
disease organisms), and those which are outside the target area (such as
typical adjacent crop plants, desirable ornamentals, garden plantings, im-
portant wildlife food and cover species, and forestry, lumber, and con-
servation plantings and endangered and threatened plant species). Series
850 addresses plant toxicity with respect to that resulting from either direct
exposure (i.e. application of a pesticide to a plant) or from indirect expo-
sure (i.e. exposure resulting from movement of the pesticide through the
environment as from runoff, soil erosion, spray drift, etc.).
(2)	Purpose. The purpose common to all tests is to provide data
which will be used to determine the need for (and support the wording
for) precautionary labeling or other statements to minimize the potential
adverse effects to nontarget plants. Generally, the registrant will provide
adequate precautionary labeling with respect to nontarget plants such as
crops, 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 endangered or threatened plants listed by the Depart-
ment of Interior.
(3)	Organization, (i) This group of guidelines contains two broad
areas of testing procedures:
(A)	Toxicity to plants in the target area.
(B)	Toxicity to plants outside of the target area.
(ii) These data should be derived from tests and reported in a manner
which complies with the general test standards and the general reporting
requirements contained in this guideline as well as the specific standards

and reporting requirements of each guideline in OPPTS Series 850, Group
D, Nontarget Plants Test Guidelines.
(c) Definitions. Terms used in this guideline have the meanings set
forth in FIFRA at Part 162.3, section 3 regulations, and OPPTS guideline
810.1100 (for target area phytotoxicity testing). In addition, for the pur-
poses of this guideline group, the following definitions apply:
Algae includes all chlorophyllous Thallophyta other than the
Bryophyta. It includes the blue-green algae (Cyanobacterium or
Cyanophyta), green algae (Chlorophyta), golden algae and diatoms
(Crysophyta), brown algae (Phaeophyta), red algae (Rhodophyta), and
golden-green algae (Xanthophyta).
Aquatic plants includes those plants that are totally aquatic (free-float-
ing or attached, submersed and immersed) and those which are semiaquatic
such as swamp and wetland plants.
Axenic is a culture of Lemna fronds free from other organisms.
Colony is an aggregate of mother and daughter fronds attached to
each other.
Desirable plants are those plants that are not to be detrimentally af-
fected during pesticide application. They may include crops, ornamentals,
or wild plants inside or outside of the area of intended application.
ECX is the external pesticide concentration required to cause a det-
rimental change or alteration (in a nontarget plant) expressed as a percent
(X) in comparison to untreated control plants. EC05, EC25, and EC50
are the concentrations required to effect a 5, 25, and 50 percent detrimental
change, respectively, on nontarget plant growth or activity.
Endpoints is a measurement during or at the end of a test, or cal-
culated from test data, that may be used to assess the effects of a pesticide
on the test organism such as numbers of organisms that survive, percent
emergence, visual phytotoxicity, growth rate measurements like plant
height, plant dry weight, root dry weight.
Frond is a single Lemna leaf-like structure.
Frond mortality are dead fronds which may be identified by a total
discoloration (yellow, white, black, or clear) of the entire frond.
LOEC is the lowest test concentration of a material used in this test
that has an adverse effect.
Microorganism is any of those organisms classified as algae, fungi
(Myxomycota and Eumycota), and bacteria (Schizomycota).

NOEC is the highest test concentration of a material used in this test
that does not have an adverse effect.
Nontarget plant and nontarget microorganism are any plant and
microorganism species not considered to be pests in the location in which
they are growing. These species are not intended to be controlled, injured,
killed, or detrimentally-affected in any way by a pesticide. Nontarget
plants include desirable or pest host plants such as crops or ornamentals
within the target area, and desirable plants outside the target area.
Pest-free is as free of pests as reasonably possible. For all pesticide
phytotoxicity tests, damaging insects and surrounding weeds should be
controlled so that healthy desirable plants are available for testing. With
this action detrimental effects can be attributed to the pesticide in question,
not to another pesticide, or to weeds, or damaging insects.
Phytotoxicity or plant toxicity are unwanted detrimental deviations
from the normal pattern of appearance, growth, and function of plants in
response to pesticides and to other toxic chemicals that may be applied
with the pesticide. The phytotoxic response may occur during germination,
growth, differentiation, and maturation of plants, and may be of a tem-
porary or long-term nature. Phytotoxic responses include adverse effects
on growth habit, yield, and quality of plants or their commodities to the
extent that a relationship between cause and effect can be established.
Plants comprise vascular and nonvascular plants, algae, and fungi.
Representative end-use product is a pesticide product that is rep-
resentative of a major formulation category (e.g. emulsifiable concentrate,
granular product, wettable powder) and pesticide group (e.g. herbicide,
fungicide, insecticide, etc.) and contains the AI of the applicant's product.
Semiaquatic plants are plants living in transition areas between aquat-
ic and dry land areas, e.g., swamps, wetlands.
Static-renewal test is a test method in which the test solution is peri-
odically replaced at specific intervals during the test.
Target area is the area intentionally treated with a pesticide when
label use directions are followed.
Target area plants are all plants located within the target area, and
includes both desirable and undesirable species.
TEP is a typical end-use product.
TGAI is a technical grade active ingredient.
Terrestrial plants are plants that do not require saturated soils for

(d) Nontarget area phytotoxicity testing—(1) Data requirements.
Data concerning the determination of outdoor pesticidal effects on
nontarget area plants are required for use in ecological risk assessment.
(See 40 CFR 158.150.) These data are also of use in assessments of poten-
tial off-target injury to endangered and threatened plant species listed by
the Fish and Wildlife Service, Department of Interior, and when
phytotoxicity concerns arise from incidents or during Special Review.
(2)	Testing scheme. Tests in the lower tiers (Guidelines 850.4100
and 850.4400 for Tier I and 850.4200 and 850.4400 for Tier II) are de-
signed to screen chemicals to determine the potential to cause adverse af-
fects on seedling emergence, vegetative vigor, and aquatic plant growth
and reproduction. The minimal phytotoxicity data set in Tier I applies to
registrations of all pesticides except herbicides, desiccants, defoliants, and
plant regulators. These tests apply to all terrestrial, aquatic, and forestry
uses so that the Agency can assess the potential for short and long term
adverse impacts on terrestrial and aquatic ecosystems and systematically
evaluate each pesticide for potential adverse effects to endangered or
threatened species. Tier II provides for generation of dose-response testing
for outdoor uses of all known phytotoxicants, including, but not limited
to herbicides, desiccants, defoliants, plant growth regulators and any fun-
gicides, insecticides or other chemicals tested in Tier I which demonstrate
phytotoxicity. In addition, Tier III (Guidelines 850.4300 and 850.4450)
is designed to broaden the knowledge concerning any detrimental effects
on nontarget plants. Progression to Tier III would occur as needed to
evaluate appropriate risk mitigation methods. The criteria to proceed from
one tier to the next are given in 40 CFR 158.540.
(3)	Waivers. Waivers of specific nontarget phytotoxicity test data or
protocols may be requested. The request for waiver must address the prod-
uct application methodology, the pesticide product's biological, chemical,
and physical properties, and the known phytotoxic properties of the pes-
ticide product.
(4)	Substitutions. If the pesticide or the active ingredient (AI) of the
pesticide, e.g. herbicide or other phytotoxic pesticide, has been extensively
tested using screening tests or other evaluation systems that are similar
in intent to any tests of Tier I, the data from those tests may be submitted
in lieu of the required data. The term "extensively tested" means dose
response testing of at least the plants or plant families represented in
OPPTS 850.4100 and 850.4400 under environmental conditions suitable
to determine any phytotoxic effects. The reports should be submitted as
provided in paragraphs (c) of OPPTS 850.4100, 850.4400, 850.4200,
850.4300, and 850.4450. In addition, paragraph (h) of this guideline lists
the information to be provided regarding the plant screening data and the
documentation to be provided on testing procedures. The Agency will re-
serve the right to require testing as provided in Tier I if the submitted

test data do not prove to be adequate to assess a pesticide's phytotoxic
(e)	Target area phytotoxicity testing waiver of requirements. (1)
It has been determined that product performance test data include target
area phytotoxicity testing data (see Guideline 850.4025), and that data sub-
mittals for such testing may be waived by the authority of the Adminis-
trator, under FIFRA (U.S. Code 7, 136, 3(c)(5), for most kinds of pesticide
products. Such products generally include all pesticides whose uses result
in direct or indirect application to plants in the target area such as range-
lands and nonagricultural areas.
(2) Even though the Administrator will ordinarily waive the require-
ments for submittal of target areaa phytotoxicity test data as indicated in
paragraph (d)(1) of this guideline, the Administrator reserves 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 prod-
(f)	Basic test standards—(1) Purpose. This paragraph contains test
standards that apply to all studies in this series of guidelines. If a specific
test contains a standard on the same subject, that specific test standard
should take precedence in the performance of that particular study.
(2)	General. The experimental design, execution of the experiments,
classification of the organism, sampling, measurement, and data analysis
in support of an application for registration must be accomplished by use
of sound scientific techniques recognized by the scientific community. The
uniformity of procedures, materials, and reporting must be maintained
throughout the toxicity evaluation process. Refinements of the procedures
to increase their accuracy and effectiveness are encouraged. When such
refinements include major modifications of any test procedure or standard,
the Agency should be consulted before implementation. All references sup-
plied with respect to protocols or other test standards are provided as rec-
(3)	Personnel, (i) All testing and evaluation must be done under the
direction of personnel who have the education, training, and/or experience
to perform the testing and evaluation in accordance with sound scientific
experimental procedures.
(ii) To help assure consistency in the development of data, one person
should be responsible for each particular phase of the study.
(4)	Test Substance, (i) Use of a TEP instead of a TGAI is preferred
for all terrestrial nontarget plant phytotoxicity tests. Aquatic plant studies
may be conducted using the TEP or TGAI. If an adjuvant is recommended
on the product label, representative adjuvants must be included in the test
at the recommended dosage. The TEP selected for testing should be the

one with the highest percentage AI and/or the one most widely used.
TEP's that contain other AI's should be avoided or tested separately. The
use of TEP testing should eliminate the need for a separate solvent control
as the solvents will already be contained in the formulation. An untreated
(negative) control is still required. If a carrier, vehicle, or adjuvant is used
to dissolve, dilute, or modify the physical characteristics of the test sub-
stance for any study, it should not:
(A)	Interfere with the metabolism (degradation) of the test substance.
(B)	Alter the chemical properties of the test substance.
(C)	Produce physiological or toxic effects to plants.
(ii) In addition to or instead of data required by this guideline, the
Agency may require, after consultation with the applicant, data derived
from testing with:
(A)	The technical grade of an AI.
(B)	A contaminant or impurity of an active or inert ingredient.
(C)	A metabolite or degradation product of an active or inert ingredi-
(D)	A different pesticide formulation (TEP).
(E)	Any additional substance which enhances the phytotoxic activity
(up to and including synergistic effects) of the product for which registra-
tion is sought.
(F)	Any combination of the test substances listed.
(5) Nontarget plant test species, (i) The organism species or groups
to be tested are specified in OPPTS 850.4100 through 850.4450.
(ii)	Healthy plants must be used.
(iii)	Either cultivated crop, ornamental, or wild indigenous plants may
be used; endangered or threatened species as determined by the Endan-
gered Species Act of 1973 (Public Law 93-205) are not to be used. When
selecting plant test species other than corn, soybean, and a root crop, the
Agency encourages the use of sensitive plants other than crop plants—
weeds, native species, perennial species, etc. The Agency also encourages
testing of more than 10 species.
(iv)	Test organisms that are obtained from natural systems and which
are to be used for testing should be maintained under conditions similar
to their natural or normal cultural environment.
(v)	The population size of each replicate or treatment should be large
enough to assure meaningful results. Sample sizes should be selected

which will yield results that are statistically significant at the 90 to 95
percent level of confidence with a significance level of less than 0.10.
The sample size for each plant species in the tier tests should be of suffi-
cient size to support the 25 or 50 percent (EC25 or EC50) progression
criteria statistically.
(6)	Nontarget organism safety. While performing field tests, all nec-
essary measures should be taken to ensure that nontarget plants and ani-
mals, especially endangered or threatened species, will not be adversely
affected either by direct hazard or by impact on food supply or food chain.
(7)	Controls. Control groups are used to assure that effects observed
are associated or attributed only to the test substance exposure. In
phytotoxicity evaluations, all treated plots, plants, and commodities must
be compared directly to untreated control plots, plants, and commodities.
The appropriate control group should be similar in every respect to the
test group except for exposure to the test substance. Within a given study,
all test organisms including the controls should be from the same source.
To prevent bias, a system of random assignment of the test plants to test
and control groups is required. Where a carrier, vehicle, or adjuvant other
than water is used, appropriate experiments and controls should be in-
cluded to distinguish the possible action of the carrier, vehicle, or adjuvant.
(8)	Equipment, (i) All equipment used in conducting the test, includ-
ing equipment used to prepare and administer the test substance, and
equipment to maintain and record environmental conditions, should be of
such design and capacity that tests involving this equipment can be con-
ducted in a reliable and scientific manner. Equipment should be inspected,
cleaned, and maintained regularly, and be properly calibrated.
(ii) The application equipment used in testing products in small field
plot studies should be designed to simulate conventional farm equipment.
This can be accomplished by using the basic components of commercial
application equipment in the design of the small-plot equipment. For ex-
ample, nozzle types, sizes, and arrangements on small plot sprayers can
be identical to those used by growers on commercial ground sprayers. Sin-
gle-row commercial granular application equipment mounted on a garden
tractor for small plot trials should produce results comparable to a multiple
of such units on a large tractor. For large-scale field trials, commercial
application equipment should be used. Specific details as to descriptions
of equipment design, adjustment, and operation should be provided in test
(g) Evaluation and reporting requirements—(1) General, (i) Ex-
perimental use permits may be required for the terrestrial testing of pes-
ticides under field conditions involving more than 10 acres such as in stud-
ies described in OPPTS 850.4025 and 850.4300. A permit may be required
for aquatic field testing of pesticides of more than 1 acre.

(ii)	The report should include a detailed and accurate description of
test procedures, materials, results, and analysis of the data, a statement
of conclusions drawn from the analysis, and a tabular summary and ab-
stract of results. When they have been determined, the primary and second-
ary modes of action with respect to plant morphogenic and biochemical
levels should be reported.
(iii)	The metric system should be used in test reports. The U.S. Stand-
ard Measures may be used to preclude extensive conversion to the metric
system. The two systems cannot be mixed (e.g. grams per square feet).
(iv)	The English language must be used in all test reports. English
translations must be provided with foreign language reports.
(2) Test materials and methods—(i) Dates. Report the actual dates
of the studies including dates of initiation (planting, transplanting, and cul-
tural practices), applications, observations, and harvest.
(ii)	Laboratories. The names of the laboratories or institutions per-
forming the tests should be included.
(iii)	Personnel. Name and title of each investigator, and the name,
address, and phone number of the employer must be reported.
(iv)	Test substance. Identification of the test substance must be pro-
vided, including:
(A)	Chemical name, molecular structure, and qualitative and quan-
titative determination of its chemical composition.
(B)	Relevant properties of the substance tested, such as physical state,
pH, and stability.
(C)	General identification and composition of any vehicles (e.g.
diluents, suspending agents, and emulsifiers) or other materials used in
the testing of the substance.
(D)	Appropriate portions of this reporting requirement may be satis-
fied by cross-referencing to OPPTS Series 830 (Product Properties Test
(v)	Untreated control (check) plots. Detailed descriptions of plots
and plants used as controls for comparisons of toxic effects should be
included for each test. Untreated control (check) plots should be treated
and evaluated in the same manner as the treatment plots with respect to
other pesticides or chemical (fertilizers, etc.) and cultural practices.
(vi)	Test organisms. The description should include the identification
of the test organisms (genus, species, and cultivar or variety, as appro-
priate), rationale for selection of the species employed, and location of
plant collection areas including their physiographic data. When plant spe-

cies other than those identified for specific studies have been tested, their
degree of susceptibility to the pesticide should be included in the test re-
port. This susceptibility should be reported in terms of EC values as in
the regular test plant reports.
(vii)	Location. Geographic location, including relation to the target
sites, should be reported.
(viii)	Substrate conditions. (A) For aquatic pesticide applications,
the following physiographic conditions should be reported:
(7) Type of aquatic site, such as lake, pond, reservoir, stream, or irri-
gation ditch with flow rate (if moving water).
(2)	Size (area and depth or volume or length, width, and depth of
the treated areas, and of the whole site), as is appropriate to the type of
application and the type of target organisms.
(3)	Water quality, including pH, temperature, hardness, alkalinity or
salinity, where possible.
(4)	Turbidity (visual), conductivity (if possible), and dissolved oxygen
(for submerged plants only).
(5)	Soil texture, including that of soils along the immediate shoreline
or ditchbank and the submerged soil where the target pests are present
(with the percent organic material in the soil also reported). (Rec-
ommended methods and soil texture classifications may be found in para-
graph (i)(3) of this guideline.
(B) For terrestrial pesticide applications, the following physiographic
conditions should be included:
(7) The edaphic conditions and characterization including soil type
and texture, approximate pH and temperature, and Kd, and Kow values.
(2)	Where the presence of a fragipan or shallow bedrock may lead
to restricted leaching or soil waterflow, the depth of that restriction.
(3)	The degree and direction of slope and its orientation to the row
direction if the slope will lead to excessive runoff.
(ix)	Environmental conditions. (A) For growth chambers and labora-
tory experimentation, the light quality, light quantity (lux), air temperature,
humidity, photo- and thermoperiods, and watering schedules should be re-
(B) For greenhouse and field experiments, the approximate light
quantity (usually expressed in degree of cloudiness), high and low daily
air temperatures, relative humidity, and photoperiod (day length) should
be reported. The environmental conditions of the specific field site are

required only for the day of application. Area or specific field environ-
mental conditions may be used for long term studies. Rainfall is to be
reported for the duration of field experiments.
(x) Application—(A) General. The test substance application method
should be reported, including dosage rates, application equipment (nozzle,
orifice, pressure), time and number of applications with reference to season
and stage of growth), spray dilution, spray volume per unit area, and
(B)	Application rates. Dosages should be reported in units of AI
or acid equivalent as appropriate. Rates may be expressed as units of ingre-
dient per unit of land area to be treated, units of concentration (such an
parts per million), units per flow rate, or units of ingredient per unit vol-
ume applied to obtain a specified degree of foliage coverage (such as to
runoff). If a product is applied more than once within a year or growing
season, each rate and the interval between applications should be indicated.
If products are applied in a tank mixture or are applied serially, rates and
intervals, as appropriate, should be reported with identification and formu-
lation for each product.
(C)	Timing of applications. When the test substance, particularly a
herbicide, plant regulator, desiccant, or defoliant, is applied to any desir-
able nontarget plants within or adjacent to the target area, the stage of
growth or development of the plants at application should be described
in test reports.
(D)	Serial applications. In addition to the detrimental effects of the
pesticides, the times of application (or application interval) should be indi-
cated for each product or tank mix involved in the serial application.
(3) Observations, (i) Observations should be reported to include all
variations, either inhibitory or stimulatory, between the treated test orga-
nisms and the untreated control test organisms. Such variations may be
phytotoxic symptoms (chlorosis, necrosis, and wilting), formative (leaf and
stem deformation) effects, and/or growth and development rates. Observa-
tions should include the stage of development and dates when adverse
results occurred and subsided or recovered. Any lack of effects by the
pesticide should also be reported.
(ii)	Observations should be reported in sufficient detail to allow com-
plete evaluation of the results. This evaluation, to be performed by the
registrant, should include the degree or extent of effects exerted by the
pesticide in question for each replicate and variable.
(iii)	The detrimental or adverse effects to be considered and reported
during the observation period of terrestrial studies include:
(A) Stand or plant population.

(B)	Overall vigor of the plants expressed as height, weight, diameter,
length, or other similar aspect of growth.
(C)	Phytotoxicity or visible symptoms such as discoloration, mal-
formation, desiccation, or defoliation.
(D)	Lodging of plants.
(E)	Effect on root growth and structure.
(F)	Development delay or acceleration with respect to maturation.
(G)	Yield of the crop or commodity that is treated as compared to
those of crops or commodities of untreated check plots.
(iv)	Where pesticides are applied to aquatic systems and influence
plant growth and development in aquatic systems, the effects of that pes-
ticide on nontarget plants in the system and along the immediate border
should be evaluated and reported, including vigor of the plants,
phytotoxicity or other visible symptoms, and delay or acceleration with
respect to vegetative growth, flowering or sporulation, and maturation.
(v)	Uniform scoring procedures should be used to evaluate the observ-
able toxic responses.
(vi)	At least two methods of evaluation (such as quantitative and qual-
itative determinations) should be used in the evaluation of pesticide effects
on growth, reproduction, and yield of plants in greenhouse and controlled
chamber experiments. When direct measurements cannot be made, such
as in large field evaluations, a 0 to 100 or 0 to 10 rating scale should
be used, where 0 indicates no injury and 100 or 10 indicates a total effect
or kill produced by the test substance. An explanation of the steps of the
rating scale employed should be included with the report.
(vii)	Observation reports should include the basic data to be used for
the statistical analysis (see paragraph (g)(4) of this guideline). Such data
should include the actual values used to determine any percentages of ef-
fects. Raw data (chromatographs, field reports, and analysis data) may also
be included to substantiate the basic data that are required.
(4) Statistical analysis, (i) When test results such as efficacy,
phytotoxicity, or yield indicate adverse effects on crops and other
nontarget test organisms, statistical analysis is required in the evaluation
the responses. The statistical analysis should consist of:
(A)	The tabulation of the response data at each treatment level.
(B)	The determination of 25 or 50 percent detrimental effect levels
(e.g. EC25, EC50, as appropriate) and the 95 percent confidence limits,
where possible, for each.

(C) The estimated nondiscernible effect level. This is the level at
which there would be no significant effect on the intended yield, quality,
or aesthetics of the crop or plant which might be exposed.
(ii) Statistical analysis is also useful in evaluation of interactions re-
sulting from studies supporting tank mixtures or serial applications.
(5)	Supporting material. Copies of references or literature used in
modifying the test protocol, performing the test, making and interpreting
observations, and compiling and evaluating the results should be submit-
ted. Copies of unpublished literature should also be included. Copies of
the recommended literature referenced in these guidelines are not required.
(6)	Special test requirements. In addition to the data required in
this guideline, 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 problems, 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 guide-
(h)	Reporting elements for acceptability. (Further details are pro-
vided in each guideline.)
(1) Information to be provided regarding the nontarget plant
phytotoxicity screening data:
(i)	Mode of action (if available).
(ii)	Common and Latin names of species tested.
(iii)	Species should be tested with a minimum of five doses bracketing
NOEC and EC50 (or effect at maximum label rate for species not respond-
(iv)	Calculation of a dose-response curve with NOEC, EC05, EC25,
EC50, slope, and CI (confidence interval) for each species.
(v)	Raw data preferably in electronically readable form.
(2) Documentation to be provided on testing procedures.
(i)	Application method (ppi, pre-, post-emergence).
(ii)	Test substance and doses used (AI, end-use product, adjuvant
(iii)	Indoor vs. outdoor trials.
(iv)	Number of replicates per dose (minimum of three).
(v)	Number of plants per dose (number of plants per pot).

(vi)	Endpoints used (definition of rating scales, quantitative or quali-
tative precision).
(vii)	Seed source, stage of the plant life cycle (seed, seedling, leaf
(viii)	Date and duration of testing, soil type.
(ix)	Bottom vs. top watering and frequency of watering.
(x)	Any other relevant information pertinent to the evaluation.
(i) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1)	Boutin, C. et al. Proposed Guideline for Registration of Chemical
Pesticides: Nontarget plant testing and evaluation. Technical Report Series
No. 145, Canadian Wildlife Service, Environment Canada, pp. 1 - 91
(2)	Truelove, B., (ed). Research Methods in Weed Science. Southern
Weed Science Society. Auburn Printing Inc., Auburn, AL (1977)
(3)	U.S. Department of Agriculture. Soil Survey Manual, Handbook
No. 18 (1951).