United States

Environmental Protection 

Agency

Office of Chemical Safety

and Pollution Prevention

(7101)

EPA 712-C-020

January 2012

Ecological Effects

Test Guidelines

OCSPP 850.3000:

Background and

Special 

ConsiderationsTests with Terrestrial

Beneficial Insects, 

Invertebrates and 

Microorganisms
i

NOTICE

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.regulations.gov grouped by Series under Docket ID #s: EPA-HQ-OPPT-2009-

0150 through EPA-HQ-OPPT-2009-0159, and EPA-HQ-OPPT-2009-0576.
Page 1 of 23

OCSPP 850.3000: Background and special considerations: tests with terrestrial 

beneficial insects, invertebrates and microorganisms.

(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 C toxicity tests with terrestrial beneficial insects, invertebrates 

and soil micoorganisms. The source materials used in developing this harmonized 

OCSPP guideline are: OPP 140-1 General Information, OPP 140-2 Definitions, OPP 140-

3 Basic standards for testing, OPP 140-4 Reporting and evaluation of results, OPP 140-5 

Special test requirements (Pesticide Assessment Guidelines Subdivision L); Pesticide 

Reregistration Rejection Rate Analysis: Ecological Effects; and background materials 

included in the specific OCSPP Series 850, Group C guidelines.

(3) General. 

(i) The OCSPP Series 850, Group C provides guidelines applicable to conducting 

laboratory toxicity tests with terrestrial beneficial insects, invertebrates and soil 

microorganisms. Field tests are designed on a case-by-case basis. The guidelines 

in OCSPP Series 850, Group C are applicable to evaluating the hazards of 

industrial chemicals and pesticides to terrestrial beneficial insects, invertebrates 

and soil microorganisms exposed directly or indirectly. Data concerning the 

effects of pesticides on terrestrial beneficial insects, invertebrates, and 

microorganisms are used in ecological risk assessment of pesticides (see 40 CFR 

part 158, paragraph (k)(29) of this guideline). These data are also of use in 

assessments of potential injury to endangered and threatened species listed by the 

Fish and Wildlife Service, Department of Interior, and when toxicity concerns 

arise from incidents or during Special Review. These data are used for both 

deterministic and probabilistic risk assessments.

(ii) Information is provided on the design and conduct of tests with terrestrial 

beneficial insects, invertebrates and soil microorganisms, emphasizing the 

importance of adequately characterizing the test substance, use of suitable 

experimental design, as well as establishing the physical and chemical conditions 

of the test system for providing a scientifically sound understanding of how the 

test substance behaves under test conditions. Also considered are 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, 

since field tests are designed on a case-by-case basis. However, the OCSPP

850.3000 guideline lists critical quality assurance and reporting standards 

common to all the guidelines in the OCSPP Series 850, Group C guidelines.

(iii) The OCSPP Series 850, Group C guidelines have generally been validated in 

formal round-robin tests or informally through repeated use.
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(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 C.

(v) The guidelines contained in OCSPP Series 850, Group C recommend specific 

procedures to be used in almost all circumstances to result in a satisfactory study 

result, while they 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 experimental 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. The 

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.

(vi) For pesticides, a tiered testing approach given in 40 CFR 158.630 for 

nontarget insect data requirements provides for greater efficiency of testing 

resources while assuring data development as warranted to meet the objectives of 

a hazard or risk assessment. To reduce or eliminate unnecessary toxicity testing 

for regulatory decision making the specific test requirements for pesticides in 40 

CFR part 158 depend upon the use pattern of the pesticide and the potential for 

exposure of terrestrial beneficial insects and invertebrates. In addition, there is a 

hierarchal or tier system which progresses from basic laboratory tests to applied 

field tests, where the results of each tier of tests should be evaluated to determine 

the potential of the pesticide to cause adverse effects, and to determine whether 

further testing is warranted to meet the objectives of the hazard or risk assessment 

(40 CFR part 202). Generally, the decision as whether to proceed to the second 

tier, or longer term higher tiered tests, is based on the potential toxicity

demonstrated in the first level tests, in conjunction with other pertinent 

information such as use pattern and environmental fate profile. For nontarget 

insects the lower tier test is designed to screen test substances to determine the 

potential to cause adverse affects on pollinators on direct contact with the test 

substance (the OCSPP 850.3020 guideline). For pesticides, a Tier I test, referred 

to as a limit test in the OCSPP 850.3020 guideline, tests a single concentration 

and compares effects observed with appropriate controls. Tier II testing for 
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pesticides includes the multiple-concentration definitive test in the OCSPP

850.3020 guideline. The multiple-concentration definitive test provides for 

generation of the dose-response curve for test substances which are known insect 

toxicants or in which Tier I testing demonstrated the test substance was an insect 

toxicant. The higher tier foliar residue test with pollinators (the OCSPP 850.3030 

guideline).determines the length of time post-application that foliar residues are 

toxic to pollinators and is conducted for test substances which are known 

toxicants and use patterns resulting in exposure of honey bees. Higher tier 

nontarget insect testing includes the Field Testing for Pollinators (the OCSPP

850.3040 guideline) and are designed on a case-by-case basis to address specific 

objectives concerning detrimental effects on nontarget insects, and are performed 

under simulated or actual field conditions. Progression to higher tier tests would 

occur on a case-by-case basis to further refine and characterize the estimate of 

nontarget insect risk.

(vii) Data on toxicity to terrestrial beneficial insects, invertebrates and 

microorganisms may also be used to evaluate the potential hazard and risk of 

industrial chemicals. When the pattern of production, use, or disposal indicates 

exposure to these terrestrial organisms, these tests are strongly recommended. 

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)(12), (k)(13), (k)(18), (k)(19), (k)(31) and (k)(32) of this guideline 

for further details). The 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.

(viii) 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 C 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); 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)(23) of this guideline). The definitions in this 

section apply to the OCSPP Series 850, Group C test guidelines and where applicable, the 

individual test guidelines contain additional or test-specific definitions.

Acclimation is the physiological or behavioral adaptation of test organisms to new 

environmental conditions associated with the test procedure.

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 

CFR 152.3).
Page 4 of 23

Acute toxicity is the discernible adverse effects (lethal or sublethal) induced in an 

organism within a short exposure period (usually not constituting a substantial portion of 

the total life cycle or life span, e.g. single dose, hours, or days).

Acute toxicity test is a comparative study in which organisms are subjected to a severe, 

short-term stimulus (test substance). The organisms, exposed to different concentrations 

of the test substance (except in a limit test), are observed for a short period usually not 

constituting a substantial portion of the total life cycle or life span. Acute exposure 

typically includes a lethal biological response of relatively quick progression. 

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, antifoaming agents, buffering compounds, drift control agents, compatibility agents, stickers 

and spreaders.

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

Chronic toxicity test is a comparative study in which organisms are exposed to different 

concentrations of the test substance generally for a relatively long period that constitutes 

a substantial, nearly complete, or complete portion of the total life cycle or life span. 

Chronic exposure typically induces a sublethal biological response of relatively slow 

progression, or which is cumulative in nature. For some chemicals with certain modesof-action, shorter-term exposure may result in chronic or latent effects, and continued or 

cumulative exposure is therefore not necessary. 

Concentration-response curve is the graphical and mathematical relationship between the 

concentration of a substance and a specific biological response produced from toxicity

tests when percent response (e.g., mortality) 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 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.

Effect concentration (ECx) is the experimentally derived concentration of a test substance

in a test matrix (e.g., soil, feed) that would be expected to cause a specified effect in x

percent (x%) of a group of test organisms under specified exposure conditions.
Page 5 of 23

Effect concentration, median (EC50) is the experimentally derived concentration of a test 

substance in a test matrix (e.g., soil, feed) 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.

Holding is the period from the time test organisms are received in the laboratory until 

they are used in testing or begin acclimation to test conditions. Holding conditions may

include quarantine, lower temperatures to minimize disease, or other conditions that are 

different from test conditions. Where holding conditions are different from test 

conditions, the test organisms should be acclimated to test conditions prior to testing to 

not stress the organisms.

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., soil, feed) 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 IC50 is the concentration of test substance that would cause a 50% 

reduction in growth from the control response.

Lethal concentration (LCx) is the experimentally derived concentration of a test substance

in a test matrix (e.g., soil, feed) that would be expected to result in mortality of x% of a 

group of test organisms under specified exposure conditions. For example, the LC25 is 

the concentration of test substance that would result in mortality of 25% of the exposed 

test population.

Lethal concentration, median (LC50) is the experimentally derived concentration of test 

substance in test matrix (e.g., soil, feed) that would be expected to result in mortality of 

50% of a group of test organisms under specified exposure conditions.

Lethal dose, median (LD50) is the experimentally derived dose of the test substance that 

would be expected to result in mortality of 50% of a population of test animals which is 

treated with a single dose under specified exposure conditions.

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 quantification (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.
Page 6 of 23

Limit test is a toxicity test performed with a single test substance concentration or dose 

and a control to establish that the value for the measurement endpoint of concern (e.g., 

LC50, LD50) is greater than the test substance concentration or dose (limit concentration 

or dose, respectively).

Lowest observed effect concentration (LOEC) is the lowest concentration of a test 

substance to which organisms are exposed under specified exposure conditions that 

causes an 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.

Lowest observed effect level (LOEL) is the lowest dose level 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 LOEL and lowest observed adverse effect level (LOAEL) have the same meaning.

Maximum acceptable toxicant concentration (MATC) is the maximum 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 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 quantitative measure above the method 

detection limit.

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, mortality (e.g., LD50, LC50), 

growth (IC25, IC50), 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 culturing and testing certain 

organisms.

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 

measurements.

Microorganism is any of those organisms classified as fungi (Myxomycota and 

Eumycota), and bacteria (Schizomycota).

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 
Page 7 of 23

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.

No observed effect level (NOEL) is the highest dose level 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 NOEL is the 

test dosage immediately below the LOEL and can only be defined in the presence of the 

LOEL. Throughout these guidelines, the terms NOEL and no observed adverse effect 

level (NOAEL) have the same meaning.

Reagent water is water that has been prepared by deionization, glass distillation, or 

reverse osmosis.

Replicate is the experimental unit within a toxicity test. It is the smallest physical entity 

to which treatments can be independently assigned.

Test substance is the specific form of a chemical substance or mixture being evaluated 

(e.g., pesticide active ingredient or formulation, or industrial chemical).

Treatment group is the set of replicate test chambers 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.

Vehicle is any agent (e.g., solvent) which facilitates the mixture, dispersion, or 

solubilization of a test substance with a carrier (e.g., dust, spray solution) 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, lighting). Construction materials and equipment that are 

toxic, may affect toxicity, or that may adsorb test substances should not be used. See 

test-specific OCSPP Series 850, Group C 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.
Page 8 of 23

(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.

(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 

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)(1), (k)(2), (k)(15), (k)(16), (k)(17), 

(k)(26), (k)(27) and (k)(28) of this guideline.

(2) Calculation of endpoints―

(i) Background. 

(A) Data generated in ecotoxicity tests with terrestrial beneficial insects, 

invertebrates and microorganisms may be of three types: 

(1) 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; 

(2) Discrete, where there is a finite number of values possible or 

there is a space on the number line between two possible values; or 

(3) Continuous, where the variable can assume a continuum of 

possible outcomes (e.g., respiration rate).

(B) These data may be analyzed using regression-based techniques or 

hypothesis-testing procedures depending on the objectives and endpoints 

of a specific test guideline. Traditionally, the results of acute toxicity tests 

have been expressed as point estimates (e.g., LC50 or LD50 for lethality, or 

EC50 or IC50 for other effects), while the results of chronic tests have been 

expressed as the results of hypothesis-testing procedures to determine the 

NOEC and LOEC (or NOEL and LOEL). Regarding terminology, the 

term ICx is more appropriately used for continuous endpoints, rather than 

ECx. For information on the advantages and disadvantages of these 

approaches, see the references in paragraphs (k)(5), (k)(8), (k)(16), (k)(17) 

and (k)(20) of this guideline. Specific test guideline objectives, either 
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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 doseresponse curve (mathematical model) and to estimate one or more specific points 

(point estimates) on the curve, such as an LD10 and LD50. 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.

(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)(1), (k)(4), (k)(6), (k)(7), (k)(9), (k)(10), (k)(11), 

(k)(14), (k)(20), and (k)(30) of this guideline). Not all methods suitable 

for continuous data are appropriate for quantal data (see paragraphs (k)(4) 

and (k)(14) of this guideline). For point estimate tests, regression-based 

methods (e.g., probit) 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., LD50) 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., binomial, 

moving average, trimmed Spearman-Karber, linear interpolation (e.g., 

Boostrap ICp)) are available (see paragraphs (k)(24), (k)(27) and (k)(28) 

of this guideline). Which of these other methods is selected is dependent 

upon the shape of the concentration-response curve, the number of 

treatments with partial mortalities (i.e., where mortality is greater than 0% 

but less than 100%), the magnitude of these mortalities, and the number of 

replicates. The method used to estimate the endpoint and, if applicable, 

the 95% confidence interval for the point estimate should be recorded.

(B) Concentration-response 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 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 
Page 10 of 23

(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 multiplecomparison tests are often appropriate for continuous data and for count 

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 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)(iv)(A) of this 

guideline) or non-parametric 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 

concentration- or 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 manyone 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 change 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 
Page 11 of 23

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.

Table 1.―Types of Errors and the Probabilities of Making Correct and Incorrect 

Decisions Based on the Results of Testing

Actual (or True) Condition:

Test Decision Outcome:

Treatment Response ≥

Control Response

Treatment Response < Control 

Response

Treatment Response ≥ Control Response Correct Decision

probability = 1- alpha (α)

Type II error (False negative)

probability = beta (β)

Treatment Response < Control Response Type I error (False positive)

probability = α

Correct Decision

probability (Power of test) = 1-β

(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)(24) of this 

guideline. Examples are specifically given for 5 and 8 replicates for a 

one-tailed test alpha (α) 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)(24) of this guideline for various low α and beta 

(β) values (i.e., α + β = 0.25). See also the references in paragraph 

(k)(9)and (k)(25) of this guideline.

(D) Limit test. In a limit test it is only necessary to ascertain that: a fixed 

standard (such as the LD50 for an acute contact) is greater than a given 

threshold; and/or the response at the limit dose or concentration does not 

differ from the control response. Only one treatment, the limit dose or

concentration, and the appropriate control(s) are tested. This is referred to 

as a limit test or maximum challenge concentration test.

(1) Fixed standard. For a fixed standard limit test, the null 

hypothesis is that the estimated limit treatment parameter (e.g.,

percent survival) is greater than or equal to the fixed threshold 

value (e.g., 50% survival). The alternative hypothesis is that the 

estimated limit parameter is less than the fixed threshold value 

(e.g., 50% survival) (Concerned with direction, where response is 

inhibition relative to the control switch hypotheses around.)

Examples of statistical approaches are one sample binomial tests or 

one sample t-tests.

(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 and weight or 
Page 12 of 23

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 rank sum test, respectively.

(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)(25) 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)(25) 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 fall 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)(25) of this guideline). However, for a 

satisfactory test in a number of the Group C guidelines, test substance

concentrations should not be below the LOD (see specific OCSPP Series 

850, Group C 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- or dose-response curve and a 

specific point response on the curve (e.g., LD50) 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 LC50 or LD50 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%. The spacing between test 
Page 13 of 23

treatments depends upon the expected slope of the concentration- or doseresponse curve, information about which can be gained during a range-finding 

test. The test treatment levels (doses or concentrations) are usually selected in a 

geometric series in which the ratio is between 1.5 and 3.2. When the objective of 

the test is to determine a regression-based estimate and sampling 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 multiple-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 between 1.5 and 3.2. A key assumption is that the response data are 

monotonic with increasing concentration or dose (i.e., the degree of 

biological effect increases as concentration or dose 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 or dose 

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 or doses be included to better 

characterize the relationship of the biological response with exposure 

concentration or dose. 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 C 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 and the test chambers randomly assigned to 

locations. The locations may be randomly reassigned during the test. Randomized block 

designs or completely randomized designs may be used. For test results to be 

satisfactory, test organisms should ideally be randomly assigned to the test chambers; 

where this is not practical impartial assignment can be used (with the exception of 

assignment intentionally according to sex). (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 random or impartial organism 

assignment to test chambers.
Page 14 of 23

(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 C guideline.

(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. The objective 

of some OCSPP Group C guideline tests includes determination of both a 

regression-based point estimate (e.g., LD50) 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 

recommended non-parameteric multiple-comparison tests can not be performed 

without at least a minimum of four 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. A control group should be similar in 

every respect to the test substance treatment groups except for exposure to the test 

substance. As described in paragraph (f)(1) of this guideline, in addition to blank (or 

negative) controls normally run, 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 α-level of 0.05, the study may be considered unacceptable.
Page 15 of 23

(e) Test substance characterization―

(1) Background information on the test substance. The information in paragraphs 

(e)(1)(i) through (e)(1)(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; etc. 

Of particular relevance are rates for processes such as hydrolysis, photolysis, and 

volatilization.

(iv) Stability and solubility as relevant, under the test conditions (see paragraph 

(e)(2) of this guideline).

(v) Physical and chemical properties and stability information for the analytical 

standard (if applicable).

(vi) Analytical method for quantification of the test substance in the feed or 

dosing solutions. Analyses are conducted with the specific media for which it 

will be used during the test, i.e. under test conditions.

(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 which are conducted under the same conditions as those occurring 

during the test. These include but are not limited to:

(A) Test matrix characteristics (e.g., growth medium, soil, dust, spray, 

etc.)

(B) Temperature, humidity, lighting, etc.

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

(D) Use of the same test containers.
Page 16 of 23

(iii) A list of recommended tests is as in paragraphs (e)(2)(iii)(A) through 

(e)(2)(iii)(D) of this guideline:

(A) Stability trials should be conducted under actual test conditions.

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

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

guideline.

(D) 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 exposure matrix (soil, growth medium, dust, 

spray, etc.) 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 documented.

(4) Analytical test substance determinations. 

(i) Media to be tested and sampling frequency to document the concentration and 

stability of the test substance throughout exposure is defined in the test-specific 

guidelines in the OCSPP Series 850 Group C guidelines.

(ii) For field tests, media and frequency of testing depends on the objective of the 

study, the stability and fate of the test substance, and is determined on a case-bycase basis.

(f) Preparation of test substances. 

(1) The preferred choice for preparation of the test substance is to use reagent water 

(deionized, distilled or reverse osmosis water), providing the test substance can be 

dissolved in water and does not readily hydrolyze. 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 or suspend 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.

(i) Preferred vehicles are specific to the test and test organisms and are listed in 

each individual guideline in the OCSPP Series 850 Group C guidelines.

(ii) If a vehicle is used to prepare the test substance, a vehicle control is also 

included in the test, in addition to the no-treatment control. The same batch of 
Page 17 of 23

vehicle used to prepare the test treatment doses or concentrations is used in the 

vehicle control. For a valid test, the selected vehicle should not affect the test 

organisms 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 test organisms.

(iii) Ideally, vehicle concentration should be kept constant in the vehicle control 

and all test treatments. If the concentration of vehicle is not kept constant, the 

highest concentration of vehicle used in any test treatment level should be used in 

the vehicle control. Limits on the amount of vehicle that can be used are given in 

each guideline in OCSPP Series 850 Group C.

(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.

(3) If the test substance is a formulated preparation, the test concentrations should be 

expressed in terms of the concentration of a.i.

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

(1) Method validation.

(i) The analytical method used to measure the amount of test substance in the 

exposure matrix (e.g., soil, growth medium, dust, spray, etc.) or stock solution 

should be validated by appropriate laboratory practices before beginning the 

definitive test. 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 (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, method 

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 the matrix being sampled. Samples should be processed 

and analyzed immediately, or handled and stored in a manner which minimizes loss of 
Page 18 of 23

test substance through microbial degradation, photodegradation, chemical reaction, 

volatilization, sorption or other processes.

(3) Analysis of test samples. Concurrent with each analysis of test samples, quality 

control (fortified) samples should be analyzed. QC samples are prepared by adding 

known amounts of the test substance to the test matrix. Minimally, one QC sample 

should be at the low end of the test concentration range and one QC 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 the 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: An indication of the 

relative health of the organisms used in the test; A demonstration that the laboratory can perform 

the test procedure in a reproducible manner over a period of time; and Information to indicate 

whether the sensitivity of a particular strain or population in use at a laboratory is comparable to 

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 guidelinespecific 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 C. 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 U.S. EPA, ASTM, APHA et al, 

etc.).

(1) Temperature. Preferably, temperature should 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.

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

one representative location.

(3) Lighting. Guidance for lighting in laboratory toxicity tests can be found in the 

reference in paragraph (k)(3) of this guideline.

(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)(1)(i) through (j)(1)(vi) of this guideline:
Page 19 of 23

(i) Test facility (name and location), test dates, and personnel.

(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 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 used 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.

(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 include all information for providing a 

complete and accurate description of test procedures and evaluation of test results. 

(i) Objectives and procedures stated in the guideline, including any changes or 

deviations or occurrences which may have influenced the results of the test.

(ii) Identification of the test substance (including source, lot or batch number, and 

purity) and known physical and chemical properties that are pertinent to the test. 

As relevant, solubility and stability of the test substance under the test conditions, 

and stability of the test substance under storage conditions if stored prior to 

analysis. It should be reported if a formulation is being tested. 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 definitive testing. If vehicles are used, the name and source of the vehicle, 

the nominal concentration of the test substance in the vehicle, and the vehicle

concentration(s) used in the test.

(iv) Information about the test organisms.
Page 20 of 23

(v) A description of the test system used in definitive and any preliminary testing. 

This includes a description of the test chambers, method of test substance

introduction, number of organisms per chamber, number of replicates per 

treatment, all environmental parameters, description of any feeding during the test 

(if applicable), including type of food, source, amount given and frequency. 

(vi) Document and submit to the Agency the preliminary test results for review 

with the study to which they apply.

(vii) Results of measurements of test substance. All analytical procedures and 

results should be described. Report all chemistry methods used in preliminary 

trials, in range-finding tests, in establishing percent purity of batches of test 

substance, or in measuring concentrations in feed, dosing solutions, or animals. 

Include in the documentation a complete description of the method so 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.

(viii) Any difficulties in maintaining constant test substance concentrations should 

be reported. If it is observed that the stability or homogeneity of the test 

substance cannot be maintained, care should be taken in the interpretation of the 

results, and note made that the results may not be reproducible.

(ix) Methods, frequency, and results of environmental monitoring performed 

during the study (temperature, lighting, etc.) and other records of test conditions.

(x) Biological observations should be reported in sufficient detail to allow 

complete independent evaluation of the results (see specific test guidelines in this 

group for a description of what should be reported).

(xi) All data developed during the study that are suggestive or predictive of toxic 

effects and all concomitant gross toxicological manifestations.

(xii) 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.

(xiii) Methods used for test chamber and treatment randomization as well as 

methods for impartial assignment of test organisms to test chambers.
Page 21 of 23

(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, 20th edition. Part 8010, Toxicity: Introduction.

(2) 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. 11.06, West Conshohocken, PA. Current edition 

approved December 10, 1996, Reapproved 2003.

(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) Bruce, R.D. and D.J. Versteeg, 1992. A statistical procedure for modeling continuous 

toxicity data. Environmental Toxicology and Chemistry 11: 1485-1491.

(5) Chapman, G.A., B.S. Anderson, A.J. Bailer, R.B. Baird, R. Berger, D.T. Burton, D.L. 

Denton, W.L. Goodfellow, M.A. Heber, L.L. McDonald, T.J. Norberg-King and P.J. 

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.

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of the Entomological Society of America 16:10-15.

(7) Daum, R.J. and W. Killcreas, 1966. Two computer programs for probit analysis. 

Bulletin of the Entomological Society of America 12:365-369.

(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 

8:263-267.

(9) Fairweather, P.G., 1991. Statistical power and design requirements for environmental 

monitoring. Australian Journal of Marine and Freshwater Research 42:555-567.

(10) Finney, D.J., 1971. Probit Analysis 3rd ed., Cambridge: London and New York. 

(11) Litchfield, J.T., Jr. and F. Wilcoxon, 1949. A simplified method of evaluating 

dose-effect experiments. Journal of Pharmacological Experimental Therapy 96:99-133.

(12) Nabholz, J.V., 1991. Environmental hazard and risk assessment under the Toxic 

Substances Control Act. Science of the Total Environment, 109/110: 649-665.

(13) 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, 
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M.A., eds., ASTM STP 1179, American Society for Testing and Materials, Philadelphia, 

PA, pp. 40 -55.

(14) 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.

(15) 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

(16) 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. 

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OECD aquatic toxicology test guidelines. Report to OECD. Paris.

(18) Smrchek, J.C., R. Clements, R. Morcock, and W. Rabert, 1993. Assessing 

ecological hazard under TSCA: methods and evaluation of data, In Environmental 

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(19) 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

(20) Stephan, C.E., 1997. Methods for calculating an LC50. 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.

(21) U.S. Environmental Protection Agency, 1982. Pesticide Assessment Guidelines

Subdivision L―Hazard Evaluation: Nontarget Insects. Office of Pesticides and Toxic 

Substances, Washington, D.C. EPA-540/9-82-019

(22) U.S. Environmental Protection Agency, 1994. Pesticides Reregistration Rejection 

Rate Analysis: Ecological Effects, EPA 738-R-94-035, Office of Prevention, Pesticides 

and Toxic Substances, December, 1994

(23) U.S. Environmental Protection Agency, 1997. Terms of Environment, Glossary, 

Abbreviations, and Acronyms, Communications, Education, and Public Affairs, EPA 

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(24) U.S. Environmental Protection Agency, 2000. Methods for Measuring the Toxicity

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(25) 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.

(26) 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, DC. EPA-821-R-02-012

(27) 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.

(28) 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, DC.. EPA-821-R-02-014.

(29) U.S. Environmental Protection Agency, Code of Federal Regulations (CFR) Title 40 

- Pesticide Programs Subchapter E―Pesticide Programs. Part 158―Data Requirements 

for Pesticides.

(30) VanEwijk, P.H. and J.A. Hoekstra, 1993. Calculation of the EC50 and its confidence 

interval when a subtoxic stimulus is present. Ecotox.icology and Environmental Safety. 

25:25-32.

(31) Zeeman, M. and J. Gilford, 1993. Ecological hazard evaluation and risk assessment 

under EPA’s Toxic Substances Control Act (TSCA): an introduction. In Environmental

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Toxicology, 2

nd Edition, G.M. Rand, ed., Taylor and Francis, Washington, DC, pp. 703-

715.