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 Microorganismsi **NOTICE**
This guideline is one of a series of test guidelines established by the United States Environmental Protection Agency’s Office of C hemical Safety and Pollution Prevention (OCSPP) for use in testing pesticides and chemical substances to develop data fo r submission to the Agency unde r 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). T o distinguish these guidelines fro m guidelines issued by other organizations, the numbering convention adopted in 1994 specifically included OPPTS as part of th e guideline’s number. A ny 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 a ccepted scientific methodologies and protocols that are intended to provide data to inform regulatory decisions under TSCA, FIFRA, and/or FFDCA. T his 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 fro m the guidelines where circumstances warrant and without prior notice. At places in this guidance, the Ag ency use s the word “should.” I n this guidance, the use of “should” with regard to an action means that the action is recommended rather than mandatory. T he procedures contained in this guideline are strongly recommended for generating the data that are the subje ct 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 Ag ency will assess them fo r appropriateness on a case

- by

- case basi s. 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 men u. 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**

**O CSPP**  **850.3 0**  **00 :**  **Background and s peci**  **al considerations: test s**  **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 Analys is: 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 benef icial 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 sp ecies 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 physic al 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

a pplicable 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 b een validated in formal round

-robin tests or informally through repeated use.Page **2** of **23** (iv) Each submitted study should meet the data quality objectives for which the test is designed. T est validity elements critical to determining the scientific soundness and acceptability of the study

have been listed for each guideline in the OCSPP Serie s 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, base d 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 s ound, 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

o f procedures, materials, and reporting should be maintained throughout the toxicity

evaluation process. Refinements of the proc edures 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 a pproach given in 40 CFR 158.630 for nontarget insect data requirements provides for greater efficiency o f testing resources while assuring data development as warranted to meet the objectives of a hazard or risk assessment. T o reduce or eliminate unnecess ary 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 additi on, 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). G enerally, 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. F or nontarget insects the lower tier test is designed to screen test substance s to determine the potential to cause adverse affects on pollinators on direct contact with the test substance (the OCSPP 850.3020 guideline). F or 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 forPage **3** of **23** 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 substance s 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 substance s which are known toxicants and use patterns resulting in exposure of honey b ees. Higher tier nontarget insect testing includes the Field Testing for Pollinators (the OCSPP 850.3040 gui deline) 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 o f the extent of toxicity, and applicable to all ty pes of chemicals. (viii) While performing field tests, all necessary measures should be taken to ensure that nontarget plants and animals, especially endangered or threatened spec ies, 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 D efinitions); 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 Acrony ms” (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 conditi ons associated with the test procedure.
Active Ingredient (a.i.) is any substance (or group of structurally s imilar substances if specified by

the Agency) that will prevent, destroy, repel or mitigate any p est, or that functions as a plant regulator, desic cant, 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 ty pically 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 o f 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 f or a relatively long period that constitutes a substantial, nearly complete, or complete portion of the total life cycle or life span. Chronic exposure ty

pically induces a sublethal biological response of relatively s low progression, or which is cumulative in nature. F or 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 (

EC x) 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 ( EC 50) is the experimentally

derived concentration of a test substance in a test matrix (e.g. , soil, fe ed) 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 include d in a pesticide product (40 CFR 152.3). Inhibition concentration ( IC x) 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. F or example, the IC 25 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 IC 50 is the concentrati on of test substance that would cause a 50% reduction in growth from the control response. Lethal concentration (

LC x) 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 o f x% of a group of test organisms under specified exposure conditions. F or example, the LC 25 is the concentration of test substance that would result in mortality o f 25% of the exposed test population. Lethal concentration, median ( LC 50) is the experimentally

derived concentration of test substance in test matrix (e.g. , soil, feed) that would be expected to result in mortality o f 50% of a group of test organisms under specified exposure conditions. Lethal dose, median (

LD 50) is the experimentally derived dose of the test substance that would be expected to result in mortality o f 50% of a population of test animals which is treated with a single dose under specified exposure conditions.
Limit of detection (LOD) is the analy ti c level below which the qualitative presence of the material is uncertain. This is ty pically 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 ty pically defined by

the lowest concentration of fortified matrix successfully analyz ed.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. , LC 50 , LD 50) 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, f or 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. Exampl es of measurement endpoints include, but are not limited to, mortality (e.g. , LD 50 , LC 50), growth (IC 25 , IC 50), etc

. E ach test

- specific guideline identifies the measurement endpoint(s) to be determined by the proscribed study . The term “measurement endpo int” is used sy nony mously 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 hy pothetical 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 (S chizomycota).
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 causePage **7** of **23**

a statistically

s ignificant adverse effect as compared to the control(s). The NOEC is the test concentration immediately b elow the LOEC and can only b e 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

s ignificant adverse effect as compared to the control(s). The NOEL is the test dosage immediately below the LOEL and can only b e defined in the presence of the LOEL. Throughout these guidelines, the terms NOEL and no observed adverse effect level (NOAE L) 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 s olution) 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 ty pe 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). C onstruction materials and equipment that are toxic, may

affect toxicity, or that may adsorb test substance s should not be used. S ee test

- specific OCSPP Series 850, Group C guidelines for identification of any aty pical facility, equipment, or supplies used in the test. C onstruction materials and equipment that are toxic, may

affect toxicity, or that may sorb test substance s should not be used.Page **8** of **23**

(2) **Mainte nance**  **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. C leaning 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, progress ion 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 ty pe of statistical procedures that will be used to evaluate the results. S ee 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 ty

pes: (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 hy pothesis

-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., LC 50 or LD 50 for lethality, or EC 50 or IC 50 for other effects), while the results of chronic tests have been expressed as the results of hy pothesis

-testing procedures to determine the NOEC and LOEC (or NOELand LOEL). Regarding terminology, the term IC x is more appropriately used for continuous endpoints, rather than EC x . F or 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. S pecific test guideline objectives, eitherPage **9** of **23** point estimate or hy

pothesis

-based endpoints or both, are identified in each specific test guideline. (ii) **Point estimates**  **and concentration** 

**- response**  **or dose** 

**- response**  **tests.** This ty pe of toxicity

test is designed to allow cal culation 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 LD 10 and LD 50 . B ecause of the normal variation in sensitivity

o f 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). F or 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. F or a point estimate (e.g., LD 50) the 95% confidenc e 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 p aragraphs (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 i s 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 f or 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 ty pe 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 hy

pothesis is that no difference exists among the meanPage **10** of **23** (or median if nonparametric) control and treatment responses. The alternative hy

pothesis is that the treatment(s) result in an adverse biological effect relative to the control sample. P arametric 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 o f 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 p arametric and nonparametric multiple comparison tests requiring no assumption about the dose

-response but which restrict comparisons of the treatments to a control. Ameasure of the sensitivity

o f 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 hy pothesis 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 ty pes of incorrect and correct decisions that can be made in a hy pothesis

-based test andPage **11** of **23** the probability

o f making these decisions are represented in Table 1. F or multiple comparison tests the Ty pe I error rate is controlled to account for multiple test comparisons. **Table 1.―Ty pes 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. E ffects 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). S ee 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 LD 50 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 hy pothesis 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 hy pothesis 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 hy potheses 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 hy pothesis is that the treatment mean (or median) response is equal to the control response mean (or median) level and the alternative hy pothesis is that the treatment mean response differs from the control response. The direction of the alternative hy pothesis depends on what is considered an adverse direction for the specific response being evaluated, such as decreased survival and weight orPage **12** of **23** increased mortality

as compared to the control response. Examples of parametric and nonparametric two

- grou p 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 b e useful for a number of statistical analys is 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 benef its include consolidating data that may b e 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) **Outl iers.**  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 analys es 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 c onduct 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 pre sumed 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., LD 50) usually c onsist of one or more control treatments and at least five test treatments which should bracket the specific point (s) of concern for the test. T o obtain a reasonably precise estimate of the LC 50 or LD 50 using probit analys is 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 testPage **13** of **23** 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 c onsists 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 s elected in a geometric series in which the ratio is between 1.5 and 3.2. Akey 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) **Lim it**  **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 b e used. F or test results to be satisfactory, test organisms should ideally b e 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 sampl e 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., LD 50) and a hy pothesis

-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 hy pothesis

-base d tests, the determination of the test

- specific number of replicates depends upon the objectives of the test, the statistical method(s) that may b e 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 c an 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 ) **C ontrol** **s .**  Control groups are used to ensure that effects observed are associated with or attributed only

to the test substance exposure. A c ontrol 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 r un, a vehicle control (solvent control) is also tested if a vehicle was used to prepare the test substance

. T o demonstrate satisfactorily that the vehicle has no unacceptable effect, the highest concentration of the vehicle that was added to any

o f 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 f or 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

s ignificant difference between the control and the vehicle control, indicating either a positive or negative vehicle effect, for any o f 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 inf ormation 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

o f 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 hydrolys is, 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 analy tical 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

o f 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 analys is methods as detailed in paragraph (g) of this guideline. (D) Storage stability

of the test substance in the samples to be collected for chemical analys es should be determined. This includes determining whether and how samples can be stored for future analys is. (3) **Sample storage.**  If s amples of the exposure matrix (soil, growth medium, dust, spray, etc .) collected for chemical analys is 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, hydrolys is, oxidation, photolysis, reduction, sorption, or volatilization. S tability determination under storage conditions, whether it refers to storing the test substance before testing or storing samples awaiting analys is, is required by

GLPregulation. 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 o f 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**  **substance s.**  (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, vehicle s 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 vehicle s 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 o fPage **17** of **23** vehicle used to prepare the test treatment doses or concentrations is used in the vehicle control. F or 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 phys iological 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

a ppropriate laboratory practices before beginning the definitive test. An analy tical method is not acceptable if likely degradation products of the test substance give positive or negative interferences which cannot be systematically

i dentified and mathematically c orrected, 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 analys is of the test substance under the conditions of the test. Thus, quality

c ontrol (fortification) samples should be prepared at concentrations spanning the range of concentrations to be used in the definitive test, using the same procedures (vehicle s, 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. S amples should be processed and analyz ed immediately, or handled and stored in a manner which minimizes loss ofPage **18** of **23**

test substance through microbial degradation, photodegradation, chemical reaction, volatilization, sorption or other processes. (3) **Analysis of**  **test samples.**  Concurrent with each analys is of test samples, quality control (fortified) samples should be analyzed. Q C 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 samp le at the high end. Ac ontrol (zero

- level fortification) sample should also be included. T est sample recoveries may

b e corrected for inherent method bias as determined from the concurrent analys is of freshly fortified quality control samples. (h) **Reference toxicants** 
**.** Historically, reference toxicity testing has been thought to provide three ty pes 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 c an perform the test procedure in a reproducible manner over a period of time; and Information to indicate whether the sensitivity

o f a particular strain or population in use at a laboratory i s c omparable to those used in other fac ilities and how sensitivity varies over time. However, performance of control organisms over time may b e 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 o f 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. M ethods 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). T emperature 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 p ersonnel 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 reconstr uction 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 elem**  **ents.** 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

o f the test substance under the test conditions, and stability

o f the test substance under storage conditions if stored prior to analys is. 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 vehicle s 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, numb er of organisms per chamber, number of replicates per treatment, all environmental parameters, description of any f eeding during the test (if applicable), including ty pe 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 o f 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 c ontrol samples, and chromatograms from samples taken during either definitive or range

- finding tests, not of standard or samples from recovery tests. F or 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

o f the test substance cannot be maintained, care should be taken in the interpretation of the results, and note made that the results may not b e 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 c omplete 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 hy pothesis tests a measure of the sensitivity o f 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. S tandard Methods for the Examination of Water and Wastewater, 20 th edition. Part 8010, Toxicity : Introduction. (2) American Society for Testing and Materials, 2003. A STM E 1847

- 96. S tandard 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. A STM E 1733

- 95. S tandard guide for the use of lighting in laboratory testing. InAnnual 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

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- 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

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