
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 Considerations-
Tests with Terrestrial
Beneficial Insects, Invertebrates and Microorganisms

                                       
                                       
                                       
                                       
                                       
                                       
                                       
  
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  NOTICE
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  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.
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  	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.  
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  	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.
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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.
            (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 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).
      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, anti-foaming 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 modes-of-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.
      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.
      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 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.
      (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 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 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 (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 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 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 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 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 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 treatments depends upon the expected slope of the concentration- or dose-response 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.
      (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.


(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.
            (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-by-case 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 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 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 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 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:
		(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.
      	(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.

(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[th] 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.
	(6) Daum, R.J., 1970.  Revision of two computer programs for probit analysis.  Bulletin 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, 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.  
	(17) Pack, S., 1993.  A review of statistical data analysis and experimental design in 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 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. 22-39
	(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 175-B-97-001, December 1997.
	(24) U.S. Environmental Protection Agency, 2000.  Methods for Measuring the Toxicity and Bioaccumulation of Sediment-Associated Contaminants with Freshwater Invertebrates, Second Edition, EPA 600/R-99/064, March 2000.  
	(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 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. 7-21.
	(32) 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, 2[nd] Edition, G.M. Rand, ed., Taylor and Francis, Washington, DC, pp. 703-715.
