United States
Environmental Protection
Office of Chemical Safety
and Pollution Prevention , ' ' zlr""' °
(7101)      January 2012
       Ecological Effects
       Test Guidelines

       OCSPP 850.4000:
       Background and
       with Terrestrial and
       Aquatic Plants,
       Cyanobacteria, and
       Terrestrial Soil-Core


     This guideline is one of a series of test guidelines established by the United States
Environmental Protection Agency's Office of Chemical Safety and Pollution Prevention
(OCSPP) for use in testing pesticides and chemical substances to develop data for
submission to the Agency under the Toxic Substances Control Act (TSCA) (15 U.S.C. 2601,
et seq.), the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 U.S.C. 136, et
seq.), and section 408 of the Federal Food, Drug and Cosmetic (FFDCA) (21 U.S.C. 346a).
Prior to April 22, 2010, OCSPP was known as the Office of Prevention, Pesticides and Toxic
Substances (OPPTS). To distinguish these guidelines from guidelines issued by other
organizations, the numbering convention adopted in 1994 specifically included OPPTS as
part of the guideline's  number.  Any test guidelines developed after April 22, 2010 will use
the new acronym (OCSPP)  in their title.

     The OCSPP harmonized test guidelines serve as a compendium of accepted scientific
methodologies and protocols that are intended to provide data to inform regulatory decisions
under TSCA, FIFRA, and/or FFDCA. This document provides guidance for conducting the
test, and is also  used  by EPA, the public, and the companies that are subject to data
submission requirements under TSCA, FIFRA, and/or the FFDCA.  As a guidance
document, these guidelines are not binding on either EPA or any outside parties, and the
EPA may depart from  the guidelines where circumstances warrant and without prior notice.
At places in this  guidance, the Agency uses the word "should."  In this guidance, the use of
"should" with regard to an action means that the action is recommended rather than
mandatory. The procedures contained in this guideline are strongly recommended for
generating the data that are the subject of the guideline, but EPA recognizes that departures
may be appropriate in specific situations. You may propose alternatives to the
recommendations described in these guidelines, and the Agency will assess them for
appropriateness on a  case-by-case basis.

     For additional information about these test guidelines and to access these guidelines
electronically, please go to http://www.epa.gov/ocspp and select "Test Methods &
Guidelines" on the left side navigation menu.  You may also access the guidelines in
http://www.requlations.qov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-
0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.

OCSPP 850.4000: Background and special considerations: tests with terrestrial
and aquatic plants, cyanobacteria, and terrestrial soil-core microcosms.

(a) Scope—

       (1) Applicability. This guideline is intended to be used to help develop data to submit to
       EPA under the Toxic Substances Control Act  (TSCA) (15 U.S.C. 2601, et seq.),  the
       Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.), and
       the Federal Food, Drug, and Cosmetic Act (FFDCA) (21 U.S.C. 346a).

       (2) Background.  This guideline provides general information applicable to  conducting
       OCSPP  Series  850, Group D  toxicity tests with  terrestrial and  aquatic  plants and
       cyanobacteria (formerly referred to as blue-green algae).  The source materials used in
       developing this harmonized OCSPP test guideline include OPP 120-1  Overview, 120-2
       Definitions,  120-3 Basic Test Standards, and 120-4 General Evaluation and Reporting
       Requirements, Pesticide  Assessment Guidelines Sub-division  J—Hazard Evaluation:
       Nontarget Plants); ASTM E 1963-02, Standard Guide for Conducting Terrestrial Plant
       Toxicity Tests, the Pesticides Reregi strati on Rejection  Rate Analysis: Ecological Effects
       report;  and  the background materials in the OCSPP Series 850, Group  D  specific

       (3) General.

             (i) The OCSPP Series 850, Group D provides guidelines applicable to  conducting
             laboratory   and  field   toxicity  tests   with   terrestrial  and   aquatic   plants,
             cyanobacteria,  and terrestrial  (soil-core)  microcosms.    Where appropriate,
             individual  guidelines in  the OCSPP Series  850, Group D are  harmonized with
             OECD test guidelines.   The  guidelines in the OCSPP Series,  Group D  are
             applicable  to evaluating the  hazards  and risks  of industrial  chemicals and
             pesticides  to  various  terrestrial  and aquatic  plant  species and  cyanobacteria
             resulting from direct or indirect exposure. Data concerning the determination of
             outdoor pesticidal effects on plants and cyanobacteria are used in evaluating risks
             to nontarget plants in ecological risk assessment of pesticides (40 CFR part 158,
             paragraph (k)(28) of this guideline).  These data are also of use in assessments of
             potential 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.  Phytotoxicity data  are
             also  occasionally requested in  order to  assess the potential hazard of  certain
             pesticides to plants within the pesticide treatment area (target area testing).

             (ii) Information is provided on the design and conduct of tests with terrestrial and
             aquatic plants, cyanobacteria,  and terrestrial plant microcosms, emphasizing  the
             importance of adequately  characterizing the  test  substance, use of  suitable
             experimental designs, and establishing the physical and chemical conditions of
             the test system in order to provide a scientifically sound understanding of how the
             test  substance  behaves under  test  conditions.    Also  considered  are  an
             identification of the factors that can affect the test outcome  and interpretation of
             test results. This general information is primarily applicable to the guidelines for
             laboratory toxicity tests and microcosms, since  field tests are designed on a case-
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by-case basis. However, the OCSPP 850.4000 guideline lists critical elements of
quality assurance and reporting standards common to all the guidelines in OCSPP
Series 850, Group D guidelines.

(iii) The OCSPP Series 850, Group D guidelines have generally been validated in
formal round-robin tests or through repeated use.

(iv) Each  submitted study should meet the data quality objectives for which the
test is designed.   Test validity elements critical  to determining the scientific
soundness and acceptability of the study have been listed for each guideline in the
OCSPP Series 850,  Group D.

(v) Certain aspects of the OCSPP 850.1000 guideline are also relevant to toxicity
studies with aquatic plants and microorganisms (the OCSPP 850.4400 guideline,
Aquatic plant toxicity test using Lemna spp.;  the OCSPP 850.4450 guideline,
Aquatic plants field study; and the OCSPP 850.4500  guideline,  Algal toxicity;
and the OCSPP 850.4550 guideline, Cyanobacteria toxicity).

(vi) The guidelines  contained in the OCSPP Series 850, Group  D  recommend
specific procedures to be used in almost all  circumstances in order to result in a
satisfactory study result, but also provide general guidance that allows for  some
latitude, based upon study-specific circumstances.  It is  recognized that certain
problems, some of which are unavoidable, may arise both before and during
testing and provisions have thus been made in the  guidelines for dealing with
those that are commonly encountered.  These guidelines  provide  for exceptions,
while at the same time maintaining a high level of scientifically sound, state-of-
the-art guidance so  that following this guidance will  provide ecological effect
information that is scientifically defensible for its intended use, while also taking
into consideration the chemistry and environmental fate of the test  substance. For
a  satisfactory  test,  the experimental  design,  execution  of the  experiments,
classification of the organism, sampling, measurement, and data analysis should
be accomplished by use of sound scientific techniques recognized by the scientific
community.   Uniformity of  procedures,  materials, and  reporting  should  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, the Agency
should be consulted before implementation.  Also when in doubt, users of these
guidelines  should  consult  with  the  appropriate  regulatory  authorities for
clarification or additional information before proceeding.  All references supplied
with respect to protocols or other test  standards are provided as recommendations.

(vii) For pesticides, a tiered testing approach given in 40 CFR 158.540 for plant
protection data requirements provides for greater efficiency of testing resources
while assuring data development as  warranted for  hazard or risk  assessment.
Tests in the lower tiers (Tier I and Tier II) are designed to screen  test substances
to determine their potential to cause  adverse affects  on seedling emergence (the
OCSPP 850.4100 guideline), vegetative vigor (the OCSPP 850.4150 guideline),
and  aquatic  plant  and cyanobacteria  growth and  reproduction (the  OCSPP

                         Page 4 of29

850.4500 and OCSPP 850.4550 guidelines).  For pesticides, a Tier I test, referred
to as a limit test in these Group D guidelines, tests a single concentration and
compares effects observed with appropriate controls.  Tier II testing for pesticides
(multiple-concentration definitive test in these Group D guidelines provides for
generation   of  dose-response  curves  for  test  substances   which  are  known
phytotoxicants  or which  in Tier I  testing demonstrated phytotoxicity.  Tier III
plant tests  currently include  the  Terrestrial  Plants  Field  Study  (the OCSPP
850.4300 guideline); Terrestrial Soil-Core Microcosm Test (the OCSPP 850.4900
guideline);  and Aquatic Plants Field Study (the OCSPP 850.4450 guideline) and
are designed on a  case-by-case basis to  address specific  objectives concerning
detrimental  effects  on nontarget plants. Progression to Tier III would occur on a
case-by-case basis to further refine  and characterize the estimate of phytotoxicity
risk.  For the evaluation of pesticides, the criteria to proceed from one tier to the
next are given in 40 CFR 158.540.  Testing of pesticides in any of these tiers can
be expanded by conducting additional tests that are normally  specific to industrial
chemicals (e.g., the OCSPP 850. 4800 Plant Uptake  and Translocation Test;  the
Rhizobium-Legume Toxicity Test (the OCSPP 850.4600 guideline)  and adapting
them to pesticide use as needed to address specific exposure and effect concerns.

(viii) Phytotoxicity  data may also be used to evaluate the potential hazard and risk
of industrial chemicals. Phytotoxicity data are requested when there are verified
terrestrial exposures or aquatic exposures and are also occasionally requested for
industrial chemicals to assess their potential hazard to crop  plant test species.
This testing is part of the Tier I (base set)  suite of tests in the OPPT testing
scheme developed  for determining environmental effects  (see the references in
paragraphs (k)(10), (k)(ll), (k)(16), (k)(17),  (k)(30),  and (k)(31)of this guideline
for further  details).  This testing scheme is deterministic  for  the  most part,
flexible, sequential, consistent, iterative, transparent,  discriminatory  of the extent
of toxicity, and applicable to all types  of chemicals.

(ix) For industrial chemicals,  dose-response testing occurs at Tier I  to determine
as quickly   and as  efficiently  as possible,  the potential  phytotoxicity of these
chemicals during early seedling emergence and  growth of terrestrial  plants (the
OCSPP  850.4230  guideline) and  to aquatic  plants  (the  OCSPP  850.4500
guideline).   In  contrast to pesticides, the potential phytotoxicity  of industrial
chemicals is often not characterized. Thus, range-finding tests, a preliminary step
to define dose-response  testing, are  more commonly conducted than limit  or
maximum challenge tests  that use only one test concentration.  If phytotoxicity is
noted  at Tier  I multiple-concentration  definitive testing,  additional  seedling
emergence  tests with more plant species are conducted at Tier II to better delimit
this toxicity to sensitive plant groups  or under specific exposure scenarios.  For
example, if monocot species exhibit the greatest sensitivity,  additional monocots
are tested;  if a wetland monocot is  found to be the most  sensitive, additional
wetland monocots (and also dicots) are then tested. Chronic  (reproductive effect)
tests may sometimes be part of Tier II (or more commonly part of Tier III). Tier
II plant tests for industrial chemicals are designed to investigate potential chronic
cumulative  toxicity and bioconcentration  potential of industrial chemicals. Tier
III plant tests currently are the Plant Uptake and Translocation Test (the OCSPP
                          Page 5 of29

              850.4800 guideline), possibly the Terrestrial Soil-Core Microcosm  Test (the
              OCSPP  850.4900  guideline),  and the Rhizobium-Legume  Toxicity  Test (the
              OCSPP 850.4600 guideline).  Field testing is conducted at Tier IV, if necessary
              for widely distributed chemicals with high  exposure, which are toxic to plants.
              Testing  of industrial chemicals  in  any of these  tiers can  be  expanded by
              conducting  additional tests that are normally specific to  pesticides  (e.g.,  the
              OCSPP  850.4100, 850.4150, and 850.4300  guidelines) and adapting them to
              industrial chemicals as needed. These pesticide-specific tests should be viewed as
              supplements to the TSCA plant tests,  not as substitutes.  For industrial chemicals,
              the base set Tier I tests and requirement to proceed from one tier to the next are
              referenced in paragraphs (k)(10), (k)(ll), (k)(16), (k)(17), (k)(30), and (k)(31)of
              this guideline.

              (x) While performing field tests, all necessary measures  should be taken to ensure
              that nontarget plants and animals, especially endangered or threatened species,
              will not be adversely affected either by direct hazard or by impact on food supply
              or food chain.

(b) Definitions.  Terms used in the OCSPP Series 850, Group D guidelines have the meanings
set forth  in Section 3  FIFRA  regulations at  40 CFR  152.3 (Pesticide  Registration  and
Classification Procedures);  40  CFR 158.300 (Product Chemistry Definitions); 40  CFR part  160
(Good Laboratory Practice  Standards); the OCSPP 850.1000 guideline (background for aquatic
organism  testing); and in  TSCA Section 3 regulations 40 CFR part 792  (Good Laboratory
Practice Standards);  and the Agency's "Terms  of  Environment,  Glossary, Abbreviations  and
Acronyms" (see paragraph (k)(22) of this guideline). The definitions below apply to the OCSPP
Series 850 Group D test guidelines  and where applicable, the individual test guidelines contain
additional or test-specific definitions.

       Active ingredient (a.i.) is  any substance  (or group of structurally similar  substances if
       specified by the Agency)  that will prevent,  destroy, repel or mitigate any pest, or that
       functions as a plant regulator, desiccant,  or defoliant within the meaning of FIFRA (40

       Adjuvant is a subsidiary ingredient or additive in a mixture which modifies, enhances or
       prolongs  by  physical  action the  activity of  the  active  ingredient(s).   Examples of
       agricultural chemical adjuvants include but are not limited to surfactants, crop oils, anti-
       foaming agents, buffering  compounds, drift control agents, compatibility agents, stickers
       and spreaders.

       Algae includes the green algae (Chlorophyta), golden algae and diatoms (Chrysophyta),
       brown algae (Phaeophyta), and red algae  (Rhodophyta).   Organisms formerly classified
       as blue-green algae (Cyanophyta) are now classified as Cyanobacteria.

       Aquatic plants includes those plants that are totally aquatic (free-floating  or attached,
       submersed or emerged) that may inhabit still or flowing water bodies.

       Axenic is a culture of one organism free from other organisms.

       Concentration-response curve is the graphical and mathematical relationship between the
                                       Page 6  of29

concentration of a substance and a specific biological response produced from toxicity
tests when percent response (e.g., growth) values are plotted against concentration of test
substance for a  given exposure  duration.  This is also referred to as the dose-response
curve or concentration-effect curve.

Control refers  to  test organisms exposed  to  test conditions and test matrix (water,
sediment, medium, etc.) in the absence of any introduced test substance as part of the test
design for the purpose of establishing a basis  of comparison with a test substance for
known chemical or biological measurements.

Culture (noun)  refers to  the organisms which are raised on-site or maintained under
controlled conditions to produce  test organisms through reproduction.

Culture (verb) is to grow, raise, or maintain organisms under controlled conditions to
produce test organisms through reproduction.

Desirable plants are those plants that are not to be detrimentally affected during  pesticide
application.  They may include  crops, ornamentals, or native plants inside or outside of
the area of intended application.

Direct exposure refers to the direct application  of a pesticide or industrial chemical to a
plant or the location where the plant would reside.

Effect concentration (ECX) is the experimentally derived concentration of a test substance
in test matrix (e.g., water, growth medium, soil,  sediment) that would be  expected to
cause a specified effect in x percent (x%) of a group of test organisms under  specified
exposure conditions.

Effect concentration, median (ECso) is the experimentally  derived concentration of a test
substance in a test matrix (e.g., water, growth medium,  soil,  sediment) that would  be
expected to  cause a defined effect in 50% of a group of test organisms under  specified
exposure conditions.

Formulation, as used within these guidelines, is a packaged end use product (e.g., dust,
wettable powder, emulsifiable concentrate, ultra low volume,  etc} of the test substance
and may contain one or more active ingredients and one or more inert ingredients.

Germination refers to the physiological events associated with re-initiation of embryo
growth and  mobilization of reserve nutrients in seeds.  The emergence  of the seedling
radicle from the seed coat  defines the end of germination and the beginning of early
seedling growth.

Hormesis refers to a stimulatory effect on a given response variable, occurring at a low
exposure  concentration of  an otherwise  toxic test substance (e.g.,  where  the higher
exposure concentrations produce adverse effects).

Indirect exposure refers to exposure of plants or cyanobacteria resulting from movement
of the  pesticide  or industrial chemical through  the environment by runoff, soil erosion,
spray drift, etc.

                                Page 7 of29

Inert ingredient is any substance (or group of structurally similar substances if designated
by the Agency), other than an active  ingredient, which is intentionally included  in a
pesticide product (40 CFR 152.3).

Inhibition concentration (ICX)  is  the  experimentally derived  concentration of a test
substance in a test matrix  (e.g., water, growth medium, soil,  sediment) that would be
expected to cause  a given percent, x, inhibition or reduction in a non-quantal response
from the  smoothed mean control  response.  For example, the IC25 for growth is the
concentration of test substance that would cause a 25% reduction  in growth in a test
population from the control response and the ICso is the concentration of test substance
that would cause a 50% reduction in growth from the control response.

Limit of detection (LOD) is the analytic level below which the qualitative presence of the
material is uncertain.  This is typically defined by the lowest concentration producing a
signal two standard deviations above the background noise from a matrix blank sample.

Limit of quantitation (LOQ) is the analytic level below which the quantitative amount of
the material is uncertain. This is typically defined by the lowest concentration of fortified
matrix successfully analyzed.

Limit test is a toxicity test performed  with a single test substance concentration and a
control to establish that the value for the measurement endpoint of concern (e.g., ECso) is
greater than the test substance concentration (limit concentration).

Lowest observed effect concentration  (LOEC) is the lowest  concentration of a test
substance to which organisms are exposed  under  specified exposure  conditions  that
causes a statistically significant adverse effect as compared to the control(s).  Throughout
these guidelines,  the terms LOEC and lowest observed  adverse  effect concentration
(LOAEC) have the same meaning in these guidelines.

Maximum acceptable toxicant  concentration (MATC) is  the  highest concentration  at
which a test substance can be present and not be toxic to the test organism.  The MATC
lies within the range between the LOEC and  no observed effect concentration (NOEC).
Operationally, for  industrial chemicals, the MATC is defined as the geometric mean of
these values. The MATC is also referred to (in the Pre-Manufacture Notification (PMN)
program of OPPT) as the chronic value  or chronic no-effect-concentration (NEC).

Measured concentration is an analytically derived measure above the method detection

Measurement endpoint is a quantitative measurable response to a stressor that is used to
infer a measure of protection or evaluate risk to valued environmental entities.  Examples
of measurement endpoints include,  but are not limited to, number of organisms  that
survive, visual phytotoxicity, growth measurements (e.g., algal population density, plant
height, plant dry weight, etc}.  Each test-specific guideline identifies the measurement
endpoint(s) to be determined by the proscribed study. The term "measurement endpoint"
is used synonymously with the term "measures of effect."

Medium is the chemically-defined  culture solution used in microcosms  and in culturing
                               Page 8 of29

and testing certain organisms such as aquatic plants.

Method detection limit (MDL) is operationally defined as the concentration of constituent
that,  when processed  through the complete method, produces  a signal with  99%
probability that it is different from the blank.  It is computed as the standard deviation
multiplied by  the  Student's  t constant  corresponding to the  appropriate degrees of
freedom (n-1).  Thus, for seven  spiked samples prepared  at the hypothetical LOQ, the
MDL  is  3.143 times  the  standard  deviation of the mean  of the seven  replicate

Nominal  concentration  is,  for  aquatic tests, the  calculated concentration of active
ingredient that would exist if all test substance added to the test  solution was completely
dissolved and did not  dissipate in  any way.   For terrestrial tests, it is the target
concentration intended for application to test plants.

Nontarget microorganism is any microorganism species to which the pesticide is not
directly applied.   These species are not intended to be controlled, injured, killed, or
detrimentally-affected in any way by a pesticide.

No observed effect concentration (NOEC) is the highest concentration of a test substance
to which organisms are exposed under specified exposure conditions that  does not cause
a statistically significant adverse  effect as compared to the control(s).  The NOEC is the
test concentration immediately below the LOEC and can only be defined in the presence
of the LOEC.  Throughout  these guidelines, the terms  NOEC and no observed adverse
effect concentration (NOAEC) have the same meaning.

Nontarget plant is any plant species to which the pesticide is not directly applied. These
species are not intended to be controlled, injured, killed, or detrimentally-affected in any
way by a pesticide.  Nontarget plants  include desirable or undesirable plants outside of
the target area.

Pest-free  refers to being as free  of pests as  reasonably possible.  For all phytotoxicity
tests, damaging insects, pathogens, 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 test substance in question, not  to another pesticide, or to weeds,
damaging insects, or pathogens.

Phytotoxicity or plant toxicity refers to unwanted detrimental deviations from the normal
pattern of appearance, growth, and function  of plants in response to the  test substance.
The  phytotoxic  response  may  occur  during  germination,  growth,   differentiation,
maturation, and reproduction of plants, and may be of a temporary 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

Plants comprise vascular and nonvascular plants (including algae and cyanobacteria).

Reagent water is water that  has been prepared by deionization, glass  distillation, or
reverse osmosis.
                                Page 9 of29

Replicate is the experimental unit within a toxicity test.  It is the smallest physical entity
to which treatments can be independently assigned.

Solubility is the amount of chemical dissolvable in test water and is operationally defined
as the  amount  of test   substance  retained in  the  supernatant  of a  conventionally
centrifuged sample of test medium or dilution water.  This amount of test substance is
considered to represent a conservative measure of the most bioavailable fraction which
may include some colloidal  material not removed by centrifugation in addition to the
truly dissolved fraction.

Static renewal system is a static system in which the test solution is renewed at specified
intervals during the test.

Stock solution is the concentrated solution of the test substance which is  dissolved and
introduced into the dilution water or test medium.

Support medium is the matrix used to support the plant during growth.  There are three
types of support  media: natural, formulated,  and artificial  media.  Natural  support
medium is  derived entirely from a combination of natural soils found in the environment.
Formulated support medium is  derived from a combination  of both natural soils and
artificial media  (including components  such as sphagnum moss).  Artificial  support
medium is derived entirely from washed quartz sand or glass beads.

Target area is the  area intentionally treated with a pesticide when label use directions are

Target area plants are  all  plants located within  the target  area, and  includes both
desirable and undesirable species.

Test chamber is the container in which the test organisms are maintained during the test

Test substance refers to  the specific  form of a chemical  substance or mixture being
evaluated (e.g., pesticide  active ingredient or formulation, industrial chemical).

Test solution refers to the test substance and the dilution water or growth medium in
which the test substance is dissolved or suspended.

Terrestrial plants  are plants that do not require an aquatic medium for growth and may
include plants that inhabit semi-aquatic areas such as swamps or wetlands.

Treatment group refers to the set of replicates that receive the same amount (if any) of the
test substance; controls are treatment groups that receive none of the test substance.

Typical end-use product  (TEP) is a term used to convey direction to a data producer to
use a commonly used end-use product, a pesticide formulation for field or other  end use
(excludes products with  labeling that allows  use of  the  product to formulate  other
pesticide products), as the test substance.  The term  includes any physical apparatus used
to deliver or apply the pesticide if distributed or sold with the pesticide.

                               Page 10 of 29

       Vehicle is any agent which facilitates the mixture, dispersion, or solubilization of a test
       substance with a carrier (e.g., water, growth medium) used to expose the test organisms
       (40 CFR  160.3, 40 CFR 792.3).

(c) Apparatus, facilities, and equipment—

       (1) Laboratory  facilities  and equipment.  The type of facilities  and equipment for
       conducting the toxicity tests with the  organisms in this  group  of guidelines varies
       depending upon the nature of the test and the organism. In general, these toxicity tests
       use normal  laboratory glassware,  supplies and  equipment  as  well as  equipment for
       maintaining  the organisms under the test conditions and controlling the test conditions
       (e.g., temperature, humidity, and lighting).   See  specific  OCSPP  Series 850, Group D
       guidelines for identification of any atypical facility, equipment, or supplies used in the
       test.  Construction materials and equipment that are toxic, may affect toxicity, or that may
       sorb test substances should not be used.

       (2) Maintenance and reliability. All equipment used in conducting the test, including
       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  conducted  in  a  reliable and  scientific manner.
       Equipment should  be  inspected, cleaned, and maintained regularly,  and be properly
       calibrated.   All materials  that will  come in contact with the test organisms and  test
       substance should be cleaned before use.  Cleaning procedures should be appropriate to
       remove known or suspected contaminants.

       (3) Permits. Experimental use  permits may be required for the terrestrial testing of
       pesticides under field conditions involving more than  10 acres such as  in  studies
       described in  the OCSPP 850.4300 (terrestrial plant field testing) guideline. Permits may
       be required for aquatic field testing of pesticides of more than 1 acre.

       (4) Field equipment.  If relevant, the application equipment used  in testing products in
       small field  plot  studies  should  be  designed  to  simulate conventional  application
       equipment.  This can be  accomplished  by using the basic components  of commercial
       application equipment in the design of the small-plot  equipment.  For example, nozzle
       types, sizes,  and arrangements on small plot sprayers can be identical  to those used by
       growers on  commercial ground sprayers.  Single-row commercial granular application
       equipment mounted on a garden tractor for small plot  trials  should produce  results
       comparable to those from 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

(d) Experimental design and data analysis—

       (1) Design  elements.  Elements of experimental  design such as the number  of test
       treatments,  progression factor between  treatment levels,  number  of replicates,  and
       number of organisms per replicate and  per treatment are based upon the purpose of the
       test, variability expected in response measurements, and the type of statistical procedures
       that will  be  used to  evaluate the results.  See the test-specific guidelines for specific
                                      Page 11 of 29

information relating to these aspects of test design.  General principles of test design are
set forth in this guideline.  General guidance on the statistical analysis of ecotoxicity tests
can be  found in the  references in paragraphs (k)(l), (k)(4), (k)(13), (k)(14),  (k)(15),
(k)(25), (k)(26), and (k)(27) of this guideline.

(2) Calculation of endpoints—

       (i) Background—

              (A) Qualitative data.  Some of the data generated in phytotoxicity tests
              are qualitative,  such  as  ratings  based  upon  visual  symptoms  of
              phytotoxicity.   Qualitative data  such as  ratings  are  not  statistically
              analyzed for these tests but may be used to report qualitative no-effect

              (B) Quantitative data.

                     (1) Quantitative data  generated in phytotoxicity tests may be of
                     three types:

                           (a) Quantal (dichotomous), where the variable has only two
                           mutually exclusive  outcomes, (e.g., dead or  alive)—note
                           that quantal data are a special  case of discrete data;

                           (b)  Discrete, where there  is a finite number of values
                           possible or there is a space on the number line between two
                           possible  values (e.g., number  of fronds, fruits or seeds
                           produced); or

                           (c) Continuous, where the variable can assume a continuum
                           of possible outcomes (e.g., height, weight).

                     (2) These data may  be analyzed using regression-based techniques
                     or hypothesis-testing procedures depending on the objectives and
                     endpoints  of a  specific test  guideline.  For  information  on the
                     advantages  and disadvantages of  these approaches,  see  the
                     references in paragraphs (k)(7),  (k)(8), (k)(14), (k)(15), and  (k)(18)
                     of this  guideline.  Specific test guideline objectives, either point
                     estimate or hypothesis-based endpoints or both, are  identified in
                     each specific test guideline.

       (ii) Point estimates and concentration-response or dose-response tests.  This
       type of toxicity test is designed to allow calculation of a concentration- or dose-
       response curve (mathematical model) and to estimate one  or more specific points
       (point estimates) on the curve,  such as an EC25 or ECso.  Regarding terminology,
       the term ICX is more appropriately used for continuous endpoints, rather than ECX.
       Because of the normal variation in sensitivity of individuals within a group of test
       organisms, a measure of the degree of certainty in the model parameters and the
       point estimate  value(s)  should be determined.

                                Page 12 of 29

       (A) No single statistical technique is appropriate for all data sets, and the
       assumptions and requirements of each method should be known before
       using (see paragraphs (k)(l), (k)(9), (k)(12), (k)(18),  and (k)(29) of this
       guideline).  Not all methods  suitable for continuous data are appropriate
       for quantal data (see paragraphs (k)(6) and (k)(12) of this guideline). For
       point estimate tests,  regression-based  methods  that  model  the full
       concentration- or dose-response relationship and provide error estimates of
       the model parameters and point estimate(s) are desired.  The regression
       model used to fit data should be  recorded,  and the error estimates of the
       model  parameters  (e.g.,  standard error  of slope and intercept), and
       goodness-of-fit should be calculated and recorded. For  a point estimate
       (e.g., ICso) the  95% confidence interval and standard  error are calculated
       and recorded.  If data do not fit a  regression-based  model, other point
       estimator methods (e.g., binominal, moving average, trimmed  Spearman -
       Karber,  linear  interpolation  (e.g., Bootstrap  ICp))  are available  (see
       paragraphs (k)(23), (k)(26) and  (k)(27) of this guideline).   The method
       used to estimate the endpoint and, if applicable, 95%  confidence interval
       for the point estimate, should be recorded.

       (B) To account for experimental variability, select a statistical method that
       retains  data  for  the  individual  replicates  through  the  regression
       calculations as opposed to pooling the replicates.  Where hormesis occurs,
       it is  preferable  to select a statistical  method that  provides for analysis of
       the impact of  stimulation on the calculated  endpoints as  opposed  to
       discarding stimulatory data.

       (C) Concentration-effect models  are good estimating tools  only for  the
       range of concentrations  used to  fit them; therefore,  endpoints  that  are
       extrapolated  beyond the  range  of the concentrations  tested would  be
       considered to  be  of  lower  confidence or  potentially, of such  low
       confidence that they would not be appropriate to estimate.

(iii) Hypothesis-based methods—

       (A) Multiple-concentration  or  multiple-dose definitive tests.   In this
       type of test, the purpose is to determine if the biological response to a
       treatment  level differs  from the  response  of  the control.   Hypothesis
       testing-based endpoints, expressed as the NOEC and LOEC (or NOEL and
       LOEL),  are calculated by determining  statistically significant differences
       from the control.  The null hypothesis  is that no  difference exists among
       the mean (or median if nonparametric), control and treatment responses.
       The  alternative hypothesis is that the treatment(s) result in an  adverse
       biological   effect  relative to  the  control  sample.     Parametric  and
       nonparametric  analysis  of  variance  (ANOVA)  tests  and  multiple-
       comparison tests  are often appropriate for continuous data and  may  be
       appropriate for some categorical  data (rank, order, score).   Contingency
       table tests  are usually appropriate for categorical data.  Parametric tests are
       based on  normal  distribution theory and  assume that  the data within

                         Page 13 of 29

treatments  are  a  random  sample  from   an  approximately  normal
distribution  and that the  error variance is  constant  among treatments.
These assumptions should be examined using appropriate tests, and data
transformations  (see  paragraph  (d)(2)(v)(A))  of  this  guideline  or
nonparametric techniques  should be used where the assumptions  are not
met. Where possible multiple comparison tests that restrict the number of
comparisons  made  should  be used.    Generally, the  more  powerful
multiple-comparison  tests  are those  which assume  a dose-response
relationship  in the  data.  When  the  assumption of a monotonic dose-
response  holds,  Williams'  and  Jonckheere's  test,  respectively,  are
examples of parametric and nonparametric tests that can be used.  When
the assumption of a monotonic dose-response fails, Dunnett's t-test and
either Steel's  many-one rank  test or the Wilcoxon rank sum test with
Bonferroni adjustment, respectively,  are examples  of  parametric  and
nonparametric multiple comparison tests requiring no assumption about
the dose-response but which  restrict comparisons of the treatments to a
control.  A measure of the sensitivity of the test, such as the minimum
significant   difference  (parametric   tests),   should   be   calculated.
Alternatively,  a calculation of the  number of replicates necessary to
achieve data quality objectives given  the actual measured test responses
and variability should be  made.  At  a  minimum, the percent  reduction
from the control for each treatment should be calculated.

(B) Types of decisions and errors.

       (1) Table 1 presents the two possible outcomes and decisions that
       can be  reached in the statistical hypothesis  tests discussed in
       paragraph (d)(3)(ii)(A) of this guideline:

              (a) There is no difference among the mean control  and
              treatment responses; or

              (b) There is a difference among the  mean control  and
              treatment  responses  (concerned with  direction,   where
              response is adverse relative to the control).

       (2) Statistical tests of hypothesis can be designed to control for the
       chances of making incorrect decisions.  The types of incorrect and
       correct decisions that can be made in a hypothesis-based test and
       the probability of making these decisions are represented in Table
       1. For multiple comparison tests the Type I error rate is controlled
       to account for multiple test comparisons.
                  Page 14 of 29

       Table 1.—Types of Errors and the Probabilities of Making Correct and Incorrect
Decisions Based on the Results of Testing
Test Decision Outcome:
Treatment Response = Control
Treatment Response < Control
Actual (or True) Condition:
Treatment Response =
Control Response
Correct Decision
probability = 1- alpha (a)
Type I error (False positive)
probability = a
Treatment Response < Control
Type II error (False negative)
probability = beta (p)
Correct Decision
probability (Power of test) = 1-p
                    (C) Power of the test.  Power of the test versus percent reduction in
                    treatment response relative to the control mean at various coefficients of
                    variation  is  provided in  the  reference  in  paragraph  (k)(23) of  this
                    guideline.  Examples are specifically given for 5  and 8 replicates for a
                    one-tailed test alpha  (a) of  0.05 and 0.10.  Effects on the number of
                    replicates at  various  coefficients of variation  are also provided in the
                    reference in paragraph (k)(23) of this guideline for various low a and beta
                    (P) values (i.e., a + P = 0.25). See also the references in paragraphs (k)(9)
                    and (k)(24) of this guideline.

                    (D) Limit test. In a limit test it is only necessary to ascertain that: a fixed
                    point standard (such as the £€25 or IC25 for terrestrial plants or ECso or
                    ICso for aquatic plants) is greater than a given "limit" concentration (Tier I
                    test for pesticides); and the response at a given "limit" concentration does
                    not differ from  the  control response.   Only one  treatment, the  "limit"
                    concentration, and the appropriate control(s) are tested.  This is referred to
                    as a limit test or maximum challenge concentration test.

                           (1) Fixed point standard.  For a fixed point limit test, the  null
                           hypothesis is that the limit treatment mean response as compared
                           to the control response  is greater than  or equal to a fixed point
                           response  of  concern (e.g., 25%   for  terrestrial  plants).    The
                           alternative hypothesis is that the limit treatment mean response as
                           compared to the control  response is  less than  the fixed point
                           response  of concern. (Concerned with direction, where response  is
                           inhibition relative to the control switch hypotheses around.)

                           (2) Difference between two means (or medians).  For testing  if
                           the treatment level affects the test organism, the null hypothesis  is
                           that the  treatment  mean (or  median) response  is equal to the
                           control response mean (or median)  level  and the alternative
                           hypothesis is that the treatment mean response differs from the
                           control response.   The  direction   of the  alternative hypothesis
                           depends on what is considered  an adverse direction for the specific
                           response  being evaluated, such as decreased survival, shoot height
                           and biomass  or increased mortality as compared to the  control
                           response.  Examples of parametric  and nonparametric two-group
                           comparison tests are Student's t-test and Wilcoxon rank sum test,
                                     Page 15 of 29

       (iv) Transformations, outliers, and non-detects—

              (A) Transformations.  Transforma-tion of data (e.g., square root,  log,
              arcsine-square root) may be  useful for a number of statistical  analysis
              purposes.  The two main reasons are to satisfy assumptions for statistical
              testing and to derive a linear relationship between two variables, so that
              linear  regression analysis  can  be  applied.   Added benefits  include
              consolidating data that may be spread out or that have several  extreme
              values (see reference  in paragraph (k)(24) of this guideline).  Once the
              data have been transformed, all statistical  analyses are performed on the
              transformed data.

              (B) Outliers. Outliers are measurements that are extremely large or small
              relative to  the  rest  of  the data  and,  therefore,  are suspected  of
              misrepresenting the  population from which they were collected.  Unless
              there  is  a  known  documented  reason  for  the  outlier(s),   such  as
              measurement system  problems  or instrument breakdown, the statistical
              analyses performed  should at a minimum include results using the full
              data set (i.e.,  the suspected outlier(s) are not discarded).  Outliers should
              not be discarded based on  a  statistical  outlier test  (see  reference in
              paragraph  (k)(24) of this  guideline).   The  analyst  may  conduct  all
              statistical analysis of  the data with both a full and truncated  (presumed
              outliers are discarded) data set, however, so that the effect of the presumed
              outlier(s) on the conclusion may be assessed.

              (C) Nondetects.  Data generated from chemical  analysis that falls below
              the LOD  of the analytical  procedure  are  generally  described as not
              detected,  or  nondetects, (rather than as  zero or not present)  and the
              appropriate LOD should be  reported.  There are a variety of  ways to
              evaluate data that include  both detected  and non-detected values (see
              reference  in  paragraph  (k)(24) of  this  guideline).   However,  for  a
              satisfactory test  in a number of the  Group D guidelines, test substance
              concentrations should not be below the LOD (see specific OCSPP Series
              850, Group D guidelines), except in controls.

(3) Selection of test treatments—

       (i) Point estimate and concentration-response or dose-response test.  Toxicity
       tests where the objective is  the concentration-response curve and a specific point
       on the curve  (e.g.,  1C50) usually consist of one or  more control treatments and at
       least five test treatments which should bracket the  specific point(s) of concern for
       the test.  To obtain a reasonably precise estimate of the ICso using probit analysis
       for example,  one or more treatments  should be between, but not including, 0 and
       50% and one or more treatments should be between, but not including, 50 and
       100%. Where the objective is to derive the  concentration-response  curve and to
       determine more than one specific point response on the curve (e.g., IC25 and ICso),
       the use of additional treatment levels to ensure that both point response values are
       each bracketed is encouraged. The spacing between test treatments depends upon

                               Page  16 of 29

       the  expected slope of the concentration- or dose-response curve, information
       about which can be gained during a range-finding test.  The minimum geometric
       ratio between doses is 2.  The maximum geometric ratio between doses should be
       4, with adequate justification documented for using a ratio of 4.  When the
       objective of the test is to determine a regression-based estimate and sample size
       constraints apply, the use of more treatment levels is preferable to the use of more
       replicates.  The inclusion of additional treatment levels rather than additional
       replicates results in better characterization of the overall concentration- or dose-
       response relationship.

       (ii) Hypothesis-based test—

              (A)  Multiple-concentration or -dose definitive test. Each test usually
              consists of one or more control treatments and at least five test treatments
              which span the expected environmental concentrations and where at least
              the lowest treatment level  is the NOEC (or NOEL).  The test treatments
              are usually selected in a geometric series in which the ratio is minimally 2
              to 4.  A key assumption  is that the response data are  monotonic with
              increasing concentration  or dose  (i.e.,  the  degree of biological  effect
              increases as treatment concentration increases) or that there is a threshold
              response such  that a NOEC (or NOEL) for a given biological response
              should not occur at a treatment concentration higher than one found to be
              statistically different  from the control for the given biological response.
              Where these assumptions do not hold it is recommended that additional
              concentrations be included to better  characterize the relationship of the
              biological response  with   exposure  concentration.   If the  failure  is
              suspected to be due to high variability in a given response measurement,
              the number of replicates should be increased.

              (B)  Limit test.  A limit test consists of a single treatment  level and the
              appropriate control(s). Individual OCSPP  Series 850 Group D guidelines
              identify  the concentration  or dose  that satisfies the limit treatment level
              test for that guideline.

(4) Randomization. For test results to be satisfactory test treatments should be randomly
assigned to individual test chambers or the group of test chambers constituting a replicate
and the test chambers  or replicates randomly  assigned to locations. When practically
feasible, the locations should be randomly reassigned during the test. Randomized block
designs may be desirable for terrestrial plant tests, completely randomized designs may
also be used.  For test results to be satisfactory,  test  organisms (for example, seeds or
seedlings) should ideally be randomly assigned to the test containers; where this  is not
practical impartial assignment  can  be used.   (Note:  random assignment  as used here
implies a mathematically-based  unbiased  assignment  method and impartial  assignment
implies a non mathematically-based unbiased assignment procedure.) All test chambers
should be treated as  similarly as possible to eliminate potential bias in test results. The
methods used to randomize treatments among test chambers and test chambers among
locations should be recorded,  as well as methods of impartial organism assignment to test

                               Page 17 of 29

(5) Number of replicates.  The number of replicate test chambers for a given treatment
is dependent upon the objective of the specific guideline test.  Except for field tests which
are designed on a case-by-case basis, the minimum number of replicates for a given test
is  described in each individual OCSPP Series 850 Group D guideline.  The  number of
replicates selected should yield results that are statistically  significant at the  90 to 95%
level  of confidence  with a significance level of less than  0.10.  The sample size and
number of replicates for each tested plant species in the tiered test scheme should be
sufficient to calculate the IC25  or  ICso  (progression criteria for terrestrial and aquatic
plants, respectively) and to detect differences of concern  when treatments are compared
to controls.  For terrestrial plant tests, a replicate or experimental unit will usually consist
of more than one test chamber (pot or flat) because, for a satisfactory test, plants should
not be crowded in the test chambers.  For a satisfactory  test, the group of pots or flats
constituting an experimental unit or replicate should be kept together throughout the test
duration.   For tests  with algae, cyanobacteria, and aquatic vascular plants,  the test
chamber constitutes the  experimental  unit  or  replicate.  All test chambers within a
replicate and all replicates within a treatment should be treated  as similarly as possible.
Except for field tests which are designed on  a case-by-case basis, the minimum number
of replicates for a given test described in  each individual OCSPP Series 850 Group D

       (i) Regression-based  test.   When the  objective of the test is to determine a
       regression-based  estimate and sample size  constraints  apply,  the inclusion of
       additional  concentrations  rather  than  additional replicates  results in  better
       characterization of the  overall concentration-response relationship.  For pesticides
       the objective of some OCSPP Group  D guideline  tests includes determination of
       both a regression-based  point  estimate  (e.g.,  K^s) and  a   hypothesis-based
       endpoint (e.g., NOEC) in which case the minimum number of replicates will be
       determined by the hypothesis-based method.

       (ii) Hypothesis-based  test.  For hypothesis-based tests, the determination of the
       test-specific number of replicates  depends upon  the  objectives of the test, the
       statistical method(s) that  may be used,  the coefficient of variation, the  size of
       effect to be detected, and the acceptable error rate.  (Note: several of the non-
       parameteric  multiple-comparison  tests recommended   cannot  be  performed
       without at least a minimum of 4 replicates).  Individual testing facilities should
       consider variability observed in  their  laboratory  and  adjust  the  number of
       replicates upward where the minimum replication number identified  in the test
       specific  guideline is not sufficient to provide the  statistical  power to  detect
       adverse effects to the test organisms  or, if appropriate, identify and correct any
       environmental, handling,  and culturing conditions, etc. that are resulting  in the
       high variability.

(6) Controls.  Control groups  are used to ensure that effects  observed are associated with
or attributed only to the test substance exposure. In phytotoxicity evaluations, all treated
plots, plants, and commodities should be  compared  directly to untreated control plots,
plants, and commodities in order for the study to be  considered satisfactory.   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

                                Page 18 of 29

       controls should be from the same source. To prevent bias, a system of random assignment
       of the test plants to test and control groups should be used for an acceptable test. (In field
       trials, it is useful to have one set of replicates assigned, in adjacent plots, to a control and
       in order from lowest concentration to highest concentration for visual comparison, with
       the remaining replicates randomly assigned). Where a carrier, vehicle, or adjuvant other
       than water is used,  appropriate  experiments  and  controls  should be  included  to
       distinguish the possible action of the carrier,  vehicle, or adjuvant.   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.  A
       vehicle control (solvent control)  is also tested  if a vehicle was used to prepare the test
       substance.  To demonstrate satisfactorily that the vehicle has no unacceptable effect, the
       highest concentration of the vehicle that was added to any of the test chambers is used in
       the vehicle control. It is recommended that the vehicle concentration be the same at each
       treatment level.  If either the  control or vehicle control results are not satisfactory for the
       test, indicating problems with test organisms or test procedures,  the test results should be
       considered unacceptable.  If both the control and the vehicle control results verify test
       organism health and status, the control and vehicle control results are compared using an
       appropriate statistical method to determine if there is an effect of the vehicle on the test
       organisms.  If there is  a statistically significant difference between the control and the
       vehicle control, indicating either a  positive or negative vehicle effect for any of the
       measured response variables using  an  a-level of 0.05, the study may be considered

(e) Test substance characterization—

       (1) Background information on the test  substance.  The information in paragraphs
       (e)(l)(i) through (e)(l)(vi) of this guideline should be known  about the test  substance
       prior to testing:

              (i)  Chemical  name;  CAS number; molecular  structure; source; lot  or  batch
              number;  purity and/or  percent a.i.;   identities and concentrations  of major
              ingredients and  major impurities; radiolabeling if any,  location of label(s), and
              radiopurity; date of most recent assay and expiration date for sample.

              (ii) Appropriate storage and handling conditions for the test substance  to protect
              the integrity  of the test substance.   (Note: health and safety  precautions should
              also be known.  These considerations are beyond the scope of these guidelines
              and depend upon the characteristics of the test substance).

              (iii) Physical and chemical properties of the test substance, including solubility in
              water and various solvents; vapor pressure; hydrolysis at various pH;  pKa; soil
              sorption behavior, etc.  Of particular relevance are  rates for processes such as
              hydrolysis, photolysis, and volatilization.

              (iv) Stability, and if relevant, solubility,  under the test conditions (refer to OCSPP
              850.1000 guideline).

              (v)  Physical and chemical properties and stability information for the  analytical
              standard (if applicable).
                                       Page 19 of 29

       (vi) Analytical method for quantification of the test substance in the test solutions,
       test matrix (support medium) or in the dosing solutions.  Analyses are conducted
       with the specific  media  which will be used during the test; i.e.,  under test

(2) Preliminary analyses.

       (i) The Agency recommends preliminary testing  of the  test substance.   The
       information about  stability  and  solubility  of the  test  substance  should be
       developed under actual test conditions.  This information can be gained while
       doing the range-finding studies.

       (ii) Information on  the  behavior of  a test  substance  should  be based on
       experiments conducted under the same conditions as those occurring during the
       test.  These include but are not limited to:

              (A) Test matrix characteristics (e.g., support medium, culture medium,
              soil, etc}.

              (B) Temperature, humidity, lighting, etc.

              (C) With test organisms in place (when practical).

              (D) Use of the same test containers.

       (iii) The tests in paragraphs (e)(2)(iii)(A) through  (e)(2)(iii)(D) of this guideline
       should be performed:

              (A) Stability trials conducted under actual test conditions.

              (B) If relevant, solubility trials conducted under test conditions.

              (C) Chemical analysis methods as  detailed in paragraph  (g)  of this

              (D) Determination of storage stability of the test substance in the samples
              to be collected for  chemical analyses should be determined. This includes
              determining whether and how samples can be stored for future analysis.

(3) Sample storage.  If samples of the treatment solutions  or other exposure matrices
collected for chemical analysis cannot be analyzed immediately, they should be handled
and stored appropriately to minimize loss of the test substance.  Loss could be caused by
such processes as microbial degradation,  hydrolysis, oxidation,  photolysis, reduction,
sorption, or volatilization.  Stability determination under  storage conditions, whether it
refers to storing the test substance before testing or storing samples awaiting analysis, is
required by GLP regulation.  Test substance stability under storage conditions  should be
                               Page 20 of 29

       (4) Analytical test substance determinations.

              (i) For terrestrial tests, solutions to be applied should be measured at test initiation
              for a satisfactory test. See specific guidelines for any additional testing.

              (ii) For aquatic tests see the OCSPP 850.1000 guideline for guidance.

              (iii) For field tests, media and frequency of testing depends on the objective of the
              study and is determined on a case-by-case basis.

       (5) Application of test substance.  For a satisfactory study, application from equipment
       should be made from lowest concentration to highest concentration so as to minimize
       residue carryover.

       (6) Mode of action. When they have been determined, the primary and secondary modes
       of action with respect to plant morphogenic and biochemical levels should be reported.

(f) Preparation of test substances.

       (1) Procedures  for test substance preparation  for studies with algae, cyanobacteria, and
       aquatic vascular plants are  addressed  in the OCSPP 850.1000  guideline.   In terrestrial
       plant  toxicity tests,  exposure to the  test  substance is typically accomplished through
       application of the test substance on soil (or other suitable growth media matrix) or to the
       leaves and other above-ground portions of the plants.  Test substance may be added by
       weight or prepared in a concentrated stock solution.  For pesticides, the testing of typical
       end product  (TEP) in plant tests generally obviates the need for addition of a  vehicle
       since a formulation may already contain a vehicle (see paragraph (f)(3) of this guideline).

              (i) The preferred choice for preparation of a stock solution is to use reagent water
              (deionized, distilled  or  reverse osmosis  water), providing test substance can be
              dissolved in water.  If  the test substance cannot be dissolved in reagent water,
              vehicles  are often used.  If a  vehicle, i.e. a solvent,  is absolutely  necessary to
              dissolve  the test  substance, the amount used  should  not exceed the minimum
              volume necessary to dissolve the test substance.  If the  test substance is a mixture,
              formulation or commercial product,  none of the  ingredients is  considered a
              vehicle unless an extra amount is used in its preparation for testing.

              (ii) If a vehicle  is used to prepare the test substance, a vehicle control is  also
              included in  the test, in addition to the untreated (negative) control.  The same
              batch of vehicle (solvent) control used  to prepare the test treatments is used in the
              vehicle control.  For a  valid test,  the  selected vehicle should  not affect  the test
              plants at the concentration used.  A vehicle  should  not interfere  with the
              metabolism  (degradation) of the test substance, alter  the chemical properties of
              the test substance, or produce physiological or toxic effects to plants.

       (2)  All techniques used  in stock solution preparation (shaking,  stirring, sonication,
       heating, solvent, etc) should be recorded.  The appearance of the stock solution should be
       observed and recorded.

                                       Page 21 of 29

       (3) Generally, for terrestrial plant phytotoxicity testing of pesticides, the TEP is the actual
       substance tested.  The submitter should consult with the Agency if any form of the test
       substance other than TEP is to be tested. Aquatic plant studies may be conducted using
       the  TEP or technical grade of the a.i.; however, if an overspray  exposure is  used for
       emergent or floating aquatic vascular plants, the test substance should be the TEP.  If an
       adjuvant is recommended on  the  product label,  representative  adjuvants  should be
       included in the test at the recommended dosage for an acceptable test.  The TEP selected
       for testing should be the one with the highest percentage a.i. and/or the one most widely
       used.  The use of TEP testing should eliminate the need for a separate vehicle control as
       the vehicle is part of the formulation.  An untreated (negative) control is still included in
       the test.

(g) Analytical methods and sampling for verification of exposure—

       1) Method validation.

              (i) The analytical  method used to measure the test  substance should be validated
              before beginning the definitive test  by  appropriate  laboratory practices.   An
              analytical  method is not acceptable if likely degradation products of the test
              substance give positive or negative interferences which cannot be  systematically
              identified and mathematically corrected, unless it is shown  that such degradation
              products are not present in the test system  during the test.

              (ii)  Method  validation is conducted for  the purpose of determining the linear
              range, detection limit, accuracy  and precision (repeatability and reproducibility)
              of the method for analysis of the test substance under the conditions of the test.
              Thus,  quality control (fortification) samples  should be prepared at concentrations
              spanning the range  of concentrations to be used in the definitive test, using the
              same  procedures  (vehicles, etc)  and in  the same  matrix  (water,  soil, etc)
              representative of what will be used in the test.

              (iii) The method validation should include a determination of linearity between
              detector response  and test substance concentration, the LOQ, the MDL, accuracy
              (average  percent  recovery)  and precision  (relative  standard  deviation).   The
              method validation should establish the acceptance  criteria for the quality control
              (QC) samples that will be prepared and analyzed during the test.

       (2) Collection of samples. Samples should be collected in such a manner as to provide
       an accurate representation of  matrix  being  sampled.  For toxicity tests with algae,
       cyanobacteria and aquatic vascular plants, these will be aqueous samples and the OCSPP
       850.1000 guideline  should  be consulted. For terrestrial plant toxicity tests, the samples
       will generally consist of  the  dosing  solutions,  other  dosing matrix, or the  support
       medium. Analyses, if conducted, are performed to confirm the initial concentration of the
       test substance applied.   Samples should be processed and analyzed  immediately, or
       handled and stored in a manner which minimizes loss of test substance through microbial
       degradation,  photodegradation,  chemical  reaction,  volatilization,  sorption  or  other
                                      Page 22 of 29

       (3) Analysis  of test samples.  Concurrent with each analysis of test samples, quality
       control (fortified) samples should be analyzed. Quality control samples are prepared by
       adding known amounts of the test  substance to the test matrix.  Minimally, one quality
       control sample  should be at the low end of the test concentration range and one quality
       control sample at the high end. A control (zero-level fortification) sample should also be
       included.   Test sample  recoveries  may be  corrected for  inherent method bias as
       determined from concurrent analysis of freshly fortified quality control samples.

(h) Reference toxicants.  Historically,  reference toxicity testing has been thought to provide
three types of information relevant to the interpretation of toxicity test data: first, an indication of
the relative "health" of the organisms used  in the test; second, a demonstration that the laboratory
can perform  the  test procedure  in a reproducible manner over  a  period  of time; and  third,
information to indicate whether  the  sensitivity  of a particular strain or population in use at  a
laboratory is  comparable to that of those used in other facilities and how sensitivity varies over
time.  However, performance of control organisms over time may be a better indicator of success
in handling and testing of at least some organisms.   Nonetheless,  periodic reference toxicant
testing can provide  an indication of the  overall comparability of results within and among
laboratories.  Although a positive control is not standard for each test, a quarterly or semiannual
positive control  (on a guideline-specific  basis) can  serve as  a  means of detecting possible
interlaboratory or temporal variation. A reference toxicant might also be desirable when there is
any significant change in source or maintenance of test organisms or in other test conditions.

(i) Monitoring of test conditions.  Test conditions are specified in each test-specific guideline in
the OCSPP  Series 850 Group D.   These conditions include  environmental  factors such as
temperature, humidity, and lighting.  Methods used for monitoring test conditions should be in
accordance with established methods (e.g., those published by the U.S. EPA, ASTM, APHA et
al., etc. (see  paragraphs  (k)(l), (k)(2), (k)(3) of this  guideline).  Environmental conditions of
specific field  sites should be recorded daily throughout the duration of the test.

       (1) Temperature.  For greenhouse,  growth  chamber and laboratory toxicity tests or
       studies it  is  desirable that temperature be monitored continuously (recorded at least
       hourly). Alternatively, the maximum and minimum should be measured daily (which is  a
       minimum  of  at least two measurements during each 24 hour period during the study).
       Temperature  measurements should be made in at least one representative location. For
       field experiments the maximum and minimum  air temperature should be measured daily.

       (2) Humidity.  Where applicable, humidity should be monitored continuously in at least
       one representative location.

       (3) Lighting.  Guidance for lighting in greenhouse, growth chamber  and laboratory
       toxicity tests  or studies can be found in the references given  in paragraphs (k)(3), (k)(5)
       and (k)(19) of this guideline.  The photoperiod  (day length) should be  recorded and  a
       photosynthetically active  radiation  (PAR) sensor (measures light energy in the 400-700
       nanometer wavelength range) should be used  to  measure light quality in micromoles of
       photons per  square meter  per second  (|imol/m2/s).    For  field experiments, the
       approximate light quantity (usually expressed in degree of cloudiness) and photoperiod
       (day length) should be observed and recorded.  Frequency of light quality monitoring for
       a test is detailed in each individual OCSPP Series 850 Group D guideline.

                                      Page 23 of 29

       (4) Biological observations.

              (i) Where test substances are applied in the field, the effects of that test substance
              on nontarget plants in the system and along the immediate border should be
              observed and  recorded, including plant vigor, phytotoxicity  or other visible
              symptoms,   and  delay  or  acceleration  of  vegetative  growth, flowering or
              sporulation, and maturation.

              (ii)  Monitor all  variations, either inhibitory or stimulatory, between the treated
              test organisms  and the  untreated control  test  organisms for toxicity  tests
              conducted in greenhouses,  growth chambers or laboratories.  Such variations may
              be due to phytotoxicity (chlorosis, necrosis, and wilting), formative effects (leaf
              and stem deformation), and/or effects on growth or development rates.

              (iii) Uniform scoring procedures should be used to evaluate the observable toxic

              (iv) At  least two methods of evaluation (such as quantitative and qualitative
              determinations) should be used in the evaluation of test  substance effects on
              growth,  reproduction, and yield of plants in  greenhouse and controlled growth
              chamber experiments.  When direct measurements cannot be made, such as in
              large field evaluations, a O-to-100 or O-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.
(j) Reporting—
       (1) Background information. In addition to the reporting requirements prescribed in the
       Good Laboratory Practices Standards (40 CFR part 792 and 40 CFR part 160), the report
       should include the information in paragraphs (j)0)(i) through (j)0)(v) of this guideline:

             (i) Test facility (name and location),  study dates, and personnel.  If conducted
             outside of a laboratory or greenhouse, report the geographic location and describe
             the relation of this location to the occurrence  or culture of the test species in the
             surrounding area.

             (ii) The name of the sponsor, study director, principal investigator, names of other
             scientists or professionals, and the names of all supervisory personnel involved in
             the study.

             (iii) Raw data sufficient to allow independent confirmation of the study authors'
             conclusions should be presented  with the study report.  Raw data includes all
             measurements recorded  during the study  including, but not limited to,  effects
             (mortality, growth, etc),  environmental conditions (temperature,  etc) and test
             substance concentration  or dose measured as  specified and are necessary for the
             reconstruction and evaluation of the report of that study.  The absence of raw data
             may make the study incomplete and impossible to review for scientific soundness
             and thus can lead to rejection of the study as scientifically sound.
                                      Page 24 of 29

       (iv) The signed and dated reports  of each of the  individual scientists or other
       professionals involved in the study, including each  person who, at the request or
       direction of the testing facility or sponsor, conducted an analysis or evaluation of
       data or specimens from the study after data generation was completed.

       (v) The locations where all raw data and the final report are stored.

       (vi) The  statement prepared and signed by the quality assurance unit identifying
       whether  or not the study was conducted in compliance  with Good Laboratory
       Practices Standards (40 CFR part  792 or 40 CFR part  160). Alternatively the
       statement can  indicate it was  conducted under  OECD  Principles of Good
       Laboratory Practice, in accordance with the multilateral  agreement with  OECD
       member countries.

(2) Data elements. The test report should provide a complete and accurate description of
test procedures and evaluation of test results including but not limited to the material in
paragraphs (j)(2)(i) through (j)(2)(xv) of this guideline.

       (i) Objectives and  procedures stated  in  the  approved protocol,  including  any
       changes or deviations or occurrences which may have influenced the results of the

       (ii) Identification of the test substance (including source,  lot, batch number,  and
       purity), and known physical and chemical properties that are pertinent to the test.
       Provide  the  physical  state,  water solubility (if  relevant), pH,  stability  and
       degradation properties under test conditions and stability under storage conditions
       if stored prior to use  or prior to sample analysis.   Where appropriate a cross-
       reference to OCSPP  Series 830  (Product Properties  Test Guidelines) guideline
       study results can be used to report this data.

       (iii) Methods of preparation of the test substance and the concentrations or doses
       used  in limit  and definitive testing.  If vehicles  and adjuvants  (e.g. diluents,
       suspending agents, and emulsifiers) are used, the name, composition, and source
       of the vehicle and adjuvant, the nominal concentration of the test substance in the
       carrier, and the vehicle concentration(s) used in the test.

       (iv) Information about the test organisms (common  name, genus, species,  family,
       and cultivar or variety), a statement providing the  rationale for selection of the
       particular species used in the test, source, and life stage.

       (v) A description of the test system used in definitive, limit or any preliminary
       testing  this  includes  a  description  of  substrate   source,  properties  and
       characteristics;  description of the experimental unit  (test container, pots, flats,
       field  plot,  etc.), including number of test organism  per  test container and per
       experimental unit; number of replicates per treatment; controls; and method of
       test substance application including dosage rates or spray volume per unit area,
       application equipment (type,  nozzle,  orifice, pressure),  time (season, stage of
       growth) and number  of applications, tank mixture (if applicable), adjuvants (if
                                Page 25 of 29

(vi) Results of measurements of test substance.  All analytical procedures should
be  described  and  documentation provided  such  that  a bench chemist  can
independently determine what equipment to use and perform the analysis.  Also
include the raw data, standards, quality control samples, and chromatograms from
samples taken during either definitive or range-finding tests, not of standard or
samples from recovery tests.  For a satisfactory test, the accuracy of the method,
LOD, MDL, and LOQ should be given.

(vii) Exposure may be expressed as units of test substance per unit of land area
treated (e.g for terrestrial plant studies,. Ib a.i./A), units of concentration  (e.g.,
milligrams per liter for aquatic plant studies), units per flow rate,  or units of test
substance per unit volume applied to obtain a specified degree of foliage coverage
(such as to runoff).

(viii) For  pesticides, test substance concentrations or dosages should be recorded
in units of a.i. or the acid equivalent of the a.i. as appropriate.

(ix) If a product is applied more than once within a year or growing season, each
rate and the interval between applications should be recorded.

(x)  Methods,  frequency, and  results  of environmental  monitoring  performed
during  the  study  (air  temperature,   light  quality,  humidity,  photo-  and
thermoperiods, etc.) and other records of test conditions such as description of
watering schedules and any cultural practices during the test (if applicable).

(xi)  Biological observations  should be reported  in  sufficient  detail to  allow
complete  independent evaluation of the results (see specific test guidelines in this
group for specific measures of effect).   For target area applications include a
description of the stage of growth or development of nontarget plants within or
adjacent to the target area.

(xii) The stage of plant development  and study  dates  when  adverse results
occurred  and  subsided (if plants  recovered) should be recorded.  Any lack of
effects by the test substance should also be recorded.

(xiii) All data developed during the study that is suggestive or predictive of toxic
effects and all concomitant gross toxicological manifestations.

(xiv) Calculated endpoints and a description of all statistical methods, including:
software used, handling of outlier data points, handling of non-detect or zero
values, tests to validate the assumptions of the analyses, level of significance, any
data transformations, for hypothesis tests  a measure of the sensitivity of the test
(either the minimum significant difference or the percent change from the control
that this minimum difference represents). Raw data should be reported to allow
independent verification of statistical procedures.

(xv) Methods used  for  test chamber and treatment  randomization as well as
methods for random or impartial assignment of test organisms to test chambers.

                         Page 26 of 29

(k) References. The references in this paragraph should be consulted for additional background
material on this test guideline.

       (1) American Public Health  Association, American Water Works Association,  Water
       Environment Federation, 1998.  Standard Methods for the Examination of Water and
       Wastewater, 20* edition. Part 8010, Toxicity: Introduction.

       (2) American Society for Testing and Materials, 2002.  ASTM E 1963-02.  Standard
       guide for conducting terrestrial plant toxicity tests.  In Annual Book of ASTM Standards,
       Vol. 11.06, ASTM, West Conshohocken, PA.  Current edition approved December  10,

       (3) American Society for Testing and Materials, 2002.  ASTM E 1733-95.  Standard
       guide for the use of lighting in laboratory testing. In Annual Book of ASTM Standards,
       Vol. 11.06, ASTM, West Conshohocken, PA.  Current edition approved September  10,
       1995; Reapproved 2002.

       (4) American Society for Testing and Materials, 2003.  ASTM E 1847-96.  Standard
       practice for statistical analysis of toxicity tests conducted under ASTM guidelines.  In
       Annual_Book of ASTM  Standards, Vol.  11.05, ASTM, West Conshohocken, PA. Current
       edition approved December 10, 1996; Reapproved 2003.

       (5) Anon., 1981. Photosynthetically active radiation.  In: Physiological Plant Ecology.
       Lange, O.L.,  P. Nobel, B. Osmond, and H. Ziegler, eds. Vol. 12A, Encyclopedia of Plant
       Physiology. Springer-Verlag. Berlin, Heidelberg, New York.

       (6) Bruce, R.D. and DJ. Versteeg, 1992.  A statistical procedure for modeling continuous
       toxicity data. Environmental Toxicology and Chemistry 11:1485-1494.

       (7) Chapman, G.A., B.S. Anderson, AJ. Bailer, R.B. Baird, R. Berger, D.T. Burton, D.L.
       Denton, W.L. Goodfellow, M.A. Heber, L.L. McDonald,  TJ. Norberg-King  and PJ.
       Ruffier, 1996. Methods and appropriate  endpoints.  In Whole Effluent Toxicity Testing,
       D.R. Grothe,  K.L. Dickson and D.K. Reed-Judkins, eds., SETAC Press, Pensacola, FL.

       (8) deBruijn, J.H.M. and M.  Hof,  1997. How to measure no effect.  Part IV: How
       acceptable is the ECX  from an environmental policy point of view?   Environmetrics

       (9) Fairweather, P.G., 1991.  Statistical power and design requirements for environmental
       monitoring. Australian Journal of Marine Freshwater Research 42:555-567.

       (10) Nabholz, J.V.,  1991. Environmental hazard and risk  assessment under the Toxic
       Substances Control Act. Science of the Total Environment, 109/110: 649-665.

       (11) Nabholz, J.V., P. Miller and M. Zeeman,  1993.  Environmental risk assessment of
       new chemicals  under  the  Toxic Substances Control  Act (TSCA)  Section  5.   In
       Environmental Toxicology and Risk Assessment, Landis, W.G., Hughes, J.S., and Lewis,
       M.A.,  eds., ASTM STP  1179, American  Society for Testing and Materials, Philadelphia,
       PA, pp. 40 -55

                                     Page 27 of 29

(12) Nyholm, N., P.S.  Sorenson, K.O. Kusk,  and E.R.  Christensen,  1992.  Statistical
treatment  of data from  microbial  toxicity tests.   Environmental  Toxicology  and
Chemistry 11:157-167.

(13) Organization for Economic Co-operation  and Development, 1998.   Report of the
OECD Workshop on Statistical Analysis of Aquatic Toxicity Data.   OECD Series on
Testing and Assessment, No. 10. ENV/MC/CHEM(98)18

(14) Organization for  Economic Co-Operation  and Development,  2006.   Current
Approaches in the Statistical Analysis of Ecotoxicity Data: A Guidance to Application.
OECD Series on Testing and Assessment, No. 54. ENV/JM/MONO(2006)18

(15) Pack, S.,  1993.  A review of statistical data  analysis and experimental design in
OECD aquatic toxicology test guidelines. Report to OECD. Paris.

(16) Smrchek,  J.C.,  R. Clements, R.  Morcock,  and W.  Rabert, 1993.   Assessing
ecological hazard under TSCA: methods  and evaluation of data, In  Environmental
Toxicology_and Risk Assessment, Landis, W.G., Hughes, J.S., and Lewis, M.A., eds.,
ASTM STP 1179, American Society for Testing and Materials, Philadelphia, PA, pp. 22-

(17) Smrchek, J.C. and M.G. Zeeman, 1998.  Assessing risks to ecological systems from
chemicals. In Handbook of Environmental Risk Assessment and Management, P. Calow,
ed., Blackwell Science, Ltd., Oxford, UK, pp. 24-90, Chapter 3.

(18) Stephan, C.E., 1997.  Methods for calculating an LCso-  In Aquatic Toxicology and
Hazard Evaluation, ASTM  STP 634, F.L.  Mayer and J.L. Hamelink, eds., American
Society for Testing and Materials, Philadelphia,  PA.

(19) Thimijan, R.W., and R.D. Heins, 1982.  Photometric, radiometric and quantum light
units of measure: a review of procedures for interconversion. HortScience 18:818-822.

(20) U.S.  Environmental Protection  Agency, 1982.  Pesticide Assessment Guidelines
Subdivision J, Hazard Evaluation: Nontarget Plants.   Office of Pesticides and Toxics,
Washington, D.C. EPA-540/9-82-020, October  1982.

(21) U.S. Environmental Protection Agency, 1994.  Pesticides Reregi strati on Rejection
Rate  Analysis:  Ecological  Effects.   Office   of Prevention,  Pesticides  and  Toxic
Substances, Washington, D.C.  EPA 738-R-94-035.

(22) U.S. Environmental Protection Agency, 1997.  Terms of Environment, Glossary,
Abbreviations,  and  Acronyms.   Communications, Education, and  Public  Affairs,
Washington, D.C. EPA 175-B-97-001.

(23) U.S. Environmental Protection Agency, 2000.  Methods for Measuring the  Toxicity
and   Bioaccumulation   of   Sediment-Associated   Contaminants  with  Freshwater
Invertebrates, Second Edition.  Office of Water, Washington, D.C. EPA 600/R-99/064,
March 2000.
                              Page 28 of 29

(24) U.S.  Environmental Protection  Agency,  2000.   Guidance for Data  Quality
Assessment,  Practical  Methods  for  Data  Analysis.    EPA  QA/G9.    Office  of
Environmental Information, Washington, DC.  EPA/600/R-96/084, July.

(25) U.S.  Environmental  Protection Agency, 2002.  Methods for measuring the acute
toxicity of effluents and  receiving waters to freshwater  and marine organisms.  Fifth
edition, Office of Water, Washington, D.C. EPA 821-R-02-012

(26) U.S.  Environmental  Protection Agency,  2002.  Short-term methods for estimating
the chronic toxicity of  effluents and receiving waters to  freshwater organisms.  Fourth
edition, Office of Water, Washington, DC. EPA 821-R-02-013

(27) U.S.  Environmental  Protection Agency,  2002.  Short-term methods for estimating
the chronic toxicity of effluents and receiving waters to marine and estuarine organisms,
Third edition, Office of Water, Washington, DC. EPA 821-R-02-014.

(28) U.S.  Environmental  Protection Agency,  Code  of Federal Regulations (CFR) Title
40—Pesticide   Programs  Subchapter  E—Pesticide  Programs.    Part   158—Data
Requirements for Pesticides.

(29) VanEwijk, P.H. and J.A. Hoekstra, 1993.  Calculation of the ECso and its confidence
interval when a subtoxic  stimulus is present.  Ecotoxicology and Environmental Safety

(30) Zeeman, M. and J.  Gilford,  1993. Ecological hazard  evaluation and risk assessment
under EPA's Toxic Substances Control Act (TSCA): an introduction. In Environmental
Toxicology and Risk Assessment, Landis, W.G.,  Hughes, J.S., and Lewis,  M.A., eds.,
ASTM STP 1179, American  Society for Testing and Materials, Philadelphia, PA,  pp. 7-

(31) Zeeman, M.G., 1995. Ecotoxicity testing and estimation methods developed under
Section 5  of the Toxic Substances Control Act (TSCA), In Fundamentals of Aquatic
Toxicology,_2n  Edition, G.M. Rand, ed., Taylor and Francis, Washington, DC, pp. 703-
                              Page 29 of 29