
United States
Environmental Protection Agency
   Office of Chemical Safety
   and Pollution Prevention
   (7101)
EPA 712-C-015
January 2012


Ecological Effects
Test Guidelines



OCSPP 850.3200:

Soil Microbial Community Toxicity Test


                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
                                       
  
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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 850.3200: Soil microbial community toxicity test.
(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.  The source material used in developing this harmonized OCSPP test guideline is the OPPT guideline under 40 CFR 797.3700 Soil Microbial Community Toxicity Test.  This guideline was formerly Public Draft OCSPP 850.5100 (April, 1996).
(b) Purpose.  This guideline is intended for use in developing data on the toxicity of chemical substances and mixtures ("test chemicals" or "test substances") subject to environmental effects test regulations.  The guideline prescribes a test using natural soil samples to develop data on the toxicity of test substances to microbial populations indigenous to the soil.  The Environmental Protection Agency will use data from these tests to assess the hazard a test substance may present in the environment.
(c) Definitions.  The definitions in OCSPP 850.3000 apply to this guideline. The more specific definitions in this paragraph also apply:
	Ammonification is conversion of organic nitrogen compounds to ammonia (NH3) or to ammonium ion (NH4) compounds, performed by a variety of microorganisms in soil and water.
	Carbon dioxide (CO2) efflux is the evolution of CO2 gas from substrates mineralized by microbial action, indicative of respiration.
	kiloPascal (kPa) is a unit of pressure in the meter-kilogram-second system equivalent to one newton per square meter (i.e., 1 Pa x 1,000) used as a measure of water availability in soils.
	Mineralization is the complete or ultimate degradation by microorganisms of organic material to form inorganic end-products, e.g., carbon dioxide, water, chloride, ammonium, nitrates, or orthophosphate.
	Nitrification is the oxidation of ammonium salts to nitrites (NO2) and nitrates (NO3), performed by relatively specialized microorganisms.
	Surface soil is that layer of soil representing the top 15 centimeters (cm) of the area to be sampled, excluding the litter horizon.
(d) General considerations--
      (1) Summary of the test.  Surface soil is sieved and supplemented with ground, dry alfalfa.  The test substance, if soluble, is added as a solution to moisten the soil, or is added in a manner that best simulates its anticipated mode of entry in nature.  All soil samples are then incubated in darkness at approximately 22 degrees Celsius (°C).  On days 5 and 28 after introduction of the test substance, samples are analyzed for NH3 and NO3 content to establish ammonification and nitrification values, respectively, and for CO2 efflux as an indication of microbial respiration.
	(2) General test guidance.  The general guidance in OCSPP 850.3000 applies to this guideline except as specifically noted herein.
	(3) Range-finding test.  A range-finding test is usually conducted to establish if definitive testing is necessary and, if so, the concentrations of test substance to be used in the definitive test.  If the maximum concentration of test substance to which the soil microbial community is likely to be exposed in nature can be predicted, soil samples should be treated with concentrations that are 0.1, 1, and 10 times the anticipated environmental exposure concentration.  Should reasonable predictions of anticipated environmental exposure concentrations not be possible, soil samples should be exposed to a series of widely spaced concentrations of the test substance (e.g., 1, 10, 100, 1,000, 10,000 micrograms (ug) test substance per gram (g) of soil).  The test should consist of exposing at least two samples of soil from the same source to each concentration of test substance and to each control.  Two samples are the minimum number used for measurement of test endpoints.  The details of the test do not have to be the same as for definitive testing.  Treatment replication is not needed and nominal concentrations of the chemical are acceptable.  However, the range-finding test will be more useful the greater the similarity between the range-finding test and the definitive test.  Report results of range-finding tests along with the results of the definitive test, if range-finding tests are conducted.
	(4) Definitive test.  The purpose of the definitive test is to evaluate the toxicity of the test substance to the community of microorganisms residing in a particular soil and to delineate the concentration-response curve and EC50 (with 95 percent (95%) confidence intervals) for each of three variables (CO2 evolution and NH3 and NO3 soil content) that indicate the capacity of the soil microbial community to decompose organic matter and release plant nutrients.  The slopes of the concentration-response curves, associated standard errors, and the 95% confidence intervals of the slopes should also be determined.  For this determination, a minimum of five concentrations of the test chemical, plus appropriate controls, are tested.  For each soil source tested, the concentration range should be selected to define, as closely as possible, the concentration-response curve between the EC10 and EC90 for each variable.  Analytical confirmation of the test concentrations is performed as described in OCSPP 850.3000.  
(e) Test standards--
      (1) Test substance.  The substance to be tested should be technical grade, unless the test is designed to test a specific formulation, mixture, or end-use product.  OCSPP 850.3000 lists the type of information that should be known about the test substance before testing, and discusses methods for preparation of test substances.
	(2) Test duration.  The test duration is 28 days after application of the test substance. 
      (3) Test organism.  No particular species of test organisms are recommended for use in this test due to the emphasis placed on maintaining the natural state of the soil samples and their resident populations of microorganisms.  The test organisms are those that occur naturally in the soil; no other test organisms are introduced into the system.  Within a given test, all soil, including that of the controls is from the same source.  
      (4) Administration of test substance.  Depending upon the properties of the test substance, it is added via a stock solution (aqueous or solvent vehicle) to the soil, adsorbed onto the soil, or directly mixed into the soil. 
            (i) Preparation methods.  
                  (A) Reagent water should be used in making stock solutions of a water-soluble test substance.  Sufficient quantities of each concentration should be available to minimize storage time and disposal volume.  A constant volume of the stock solution should be added at the beginning of the test to each soil sample designed to receive the test substance.  OCSPP 850.3000 contains additional information on the preparation of stock solutions. 
                  (B) A test substance that is insoluble in water, but which can be suspended in an aqueous solution by a vehicle, should be added, with the vehicle, to those soil samples designated to receive the test substance.  The vehicle should be soluble in water, nontoxic to microbial life at the concentration applied, and used in a minimal amount to dissolve or suspend the test substance.  There are no preferred vehicles; however, acetone, gum arabic, ethanol, and others have been used extensively in testing herbicides, plant growth regulators, fungicides, and other chemical substances that affect plants.  Any such vehicle may be used for this test, providing it neither enhances nor inhibits the activities of the soil microbes.  Vehicle controls are included in the experimental design and tested simultaneously with the test substance, if a vehicle is used. OCSPP 850.3000 contains additional information on preparation of stock solutions using vehicles.
                  (C) A water-insoluble test substance for which no nontoxic, water-soluble vehicle is available should be dissolved in an appropriate volatile solvent.  The solution should be mixed with the ground alfalfa soil supplement, then placed in a rotary vacuum apparatus and evaporated, leaving a uniform coating of the test substance on the alfalfa.  A portion of the alfalfa is then weighed; the test substance extracted with the same organic solvent, and then the test substance is assayed before the alfalfa is mixed with the soil in the test containers.  Solvent controls (i.e., alfalfa treated only with solvent) should be included in the experimental design and tested simultaneously with the test substance.
                  (D) If the test substance is not readily soluble in water or in another commonly used vehicle, and is known to be applied or transported in nature directly to the soil as a previously prepared liquid or powder, it should be mixed, in its liquid or dry form, directly into the soil samples.  To ensure equal distribution of the test substance throughout each test sample, mix thoroughly.
            (ii) Test concentrations.  For this determination, a minimum of five treatment levels of the test chemical, plus appropriate controls, are tested.
            (iii) Soil.  To be appropriate for this guideline, a soil should possess a pH of 4 to 8, organic matter content between 1% and 8%, a cation exchange capacity (CEC) greater than 7 milliequivalents per 100 grams, and consist of less than 70% sand.  Soils to which fertilizer or pesticide(s) have been applied within the past 24 months should be avoided.  Soil collections should be restricted to the surface soil.  Large objects should be removed manually, and the remaining soil allowed to air-dry until sievable (approximately 12% water content), after which it is passed through a 2-millimeter (mm) mesh screen.  For each sample, an amount of soil equivalent to approximately 50 grams (g) oven-dry weight should be placed in an inert container.  At least one of these samples, considered to provide a baseline measurement, should be extracted immediately to determine NH3 and NO3 content (see paragraph (d)(4) of this guideline).  Alfalfa, dried and ground to pass through a 0.6-mm mesh screen should be added (0.3 g) to each remaining sample and the sample should be thoroughly mixed.  Water content of the soil should be adjusted to approximately 10 kPa by adding reagent (distilled, deionized or reverse osmosis) water containing the desired concentration of test substance (in vehicle, if necessary).  If insoluble in both water and commonly used vehicles, the test substance should be mixed into the soil as a solid and the appropriate amount of water added subsequently.  The test containers may be covered with 0.13 um (0.5 mil) polyethylene to minimize water loss, yet permit gas exchange, or left open and watered to their original weight every 7 days.  Regardless, the test substance should be applied only during the original watering.
	(5) Controls.  
            (i) Every test includes a negative control, and a vehicle (solvent) control if a vehicle is used.  Controls consist of the same soil, soil supplements, test conditions, and procedures, except that no test substance is added. 
            (ii) If an aqueous stock solution was used to prepare test concentrations, controls should receive an equal volume of reagent water without the test substance.  If a vehicle was used to dissolve or suspend the test substance, a vehicle control (i.e., solvent in water without the test substance) should also be included.  Should the test substance be a powder that is mixed directly into soil and subsequently moistened with distilled water; the control should receive an equal volume of such water only.
      (6) Number of replicates.  A total of 10 replicates of each test substance concentration, control, and vehicle control (if applicable) are used.  This provides two sets of five replicates, one set for the analyses conducted 5 days after test substance application and one set for the analyses conducted 28 days after test substance application.  The assignment of soil containers to test substance concentrations should be random.  In addition, placement of the containers in the incubation chamber should be randomized.
	(7) Facilities, apparatus and supplies -- 
            (i) Environmental chambers or incubators.  Environmental chambers or incubators should provide adequate space and controls necessary to incubate numerous soil samples in total darkness at a constant temperature for prolonged periods of time.  Chambers should be designed to prevent escape of internal air into the external environment other than through appropriate filtering material or media to prevent contamination of the external environment with the test substance.
            (ii) Test containers.  For each test, at least 60 to 70 soil containers (two series of five per concentration of test substance, two series of five for the control, and two series of five if a vehicle control is necessary) should be used.  In addition, soil to be extracted immediately as the control is most easily handled in an identical container.  All containers used in each experiment should be of equal size and volume, posses the same configuration, and should be made of the same inert material.  Wide mouth jars (for example, glass canning, 0.5 pint or 110-millilitre (mL) capacity) are adequate for this purpose.  Test containers may be covered with polyethylene film (0.13 micrometer (um); 0.5 mil) to prevent water loss.  Control containers should be handled identically to the test containers except that none of the test substance is added.
            (iii) Cleaning and sterilization.  
                  (A) Soil containers and test solution storage containers should be cleaned before use.  All equipment should be washed to remove any residues remaining from manufacturing or prior use.  A dichromate solution should not be used for cleaning containers.
                  (B) If cleaning and rinsing of previously used soil containers has been thorough, the effects of any microorganisms remaining on the interior surface of the containers should be insignificant in the presence of the new test soil.  Sterilization should not be necessary, but is considered an acceptable option.
                  (C) Soil treated with the test substance and solvent control soil should be discarded at the end of the experiment.  Disposal should conform to applicable regulations.
      (8) Environmental conditions.  Environmental conditions should be maintained as follows:
            (i) Temperature.  The temperature should be 22 °C (or that temperature to which the microorganisms are most accustomed in nature) and it should be constant within +- 2°C during the test.
            (ii) Lighting and photoperiod.  Total darkness should be used during incubation to prevent photosynthesis by algae or the growth of moss.
	
      (9) Observations -- 
            (i) Measurement of test substance.  Analytical confirmation of the concentration of test substance in the stock solutions and/or in the soil should be performed as described in OCSPP 850.3000.  Validated analytical methods are used to measure the amount of test substance in a sample before beginning the test, as described in OCSPP 850.3000. 
            (ii) Environmental conditions -- 
                  (A) Temperature.  Temperature in the incubation chamber should be monitored continuously.
                  (B) Stock solution pH.  The pH of the stock solutions should be measured before use.
		(iii) Measures of effect -- 
                  (A) Soil ammonia, nitrate and nitrogen content and carbon dioxide efflux.  Of the soil samples prepared for each test concentration or control, one set of five replicate samples should be assayed after 5 days of exposure to the test substance for NH3 and NO3 content, and then discarded.  A second set of 5 replicate samples should be analyzed the same day for CO2 evolution and then reincubated (dark, 22 °C).  On day 28 after exposure to the test substance, all remaining (reincubated) samples should be assayed first for CO2 efflux and then again for nitrogen content.
                  (B) Microorganisms.  On days 5 and 28 after introduction of the test substance, the effects of treatment should be assessed as the CO2 efflux rate and the NO3 and NH3 concentrations per gram of dry soil in treated samples, relative to untreated controls and, if applicable, vehicle controls, and to values in freshly sieved (pretreatment) soil. 
                        (1) To measure CO2 efflux, each test container is closed with a two-hole stopper fitted with Teflon tubes and twistcock connectors for attachment to the measurement apparatus.  The apparatus (see Figure 1) should deliver a stream of humid, CO2-free air to the test system, and the effluent air should be dried, diluted, and delivered for infrared gas analysis (IRGA).  The period of incubation should be adjusted to match the CO2 efflux rate with the detection capability of the IRGA, and may vary from 1 to 77 hours.  In lieu of IRGA, CO2 may be trapped in a hydroxide solution and titrated, or measured by gas chromatography.  CO2 efflux rates are expressed in terms of micrograms per gram of dry soil per hour.
	Figure 1. -- Flow Diagram for the IRGA Method of Determining CO2 Evolution from Soil Samples

                                       
                        (2) Accumulation of inorganic nitrogen is measured by extracting each soil sample with 80 mL of 1 normal potassium chloride (N KCl) solution.  After adding KCl and shaking each container by hand to suspend the soil, sample containers should be placed on a rotary shaker at high speed for 1 hour, then shaken again by hand to resuspend the soil.  Samples should be filtered (Whatman 42 low-nitrate filter paper) and the extract (filtrate) should be analyzed for NO3 and NH3.  EPA or ASTM methods should be used for these analyses.  NO3 and NH4 concentrations are expressed in terms of micrograms per gram of dry soil.
(f) Treatment of results -- 
      (1) Data summary -- 
            (i) Environmental conditions.  Temperature data should be summarized in tabular form, showing the mean, standard deviation, and range of temperature during the test.
            (ii) Carbon dioxide efflux rates, and soil nitrate and ammonia content.  CO2 efflux rates and NO3 and NH3 concentrations in treated samples and from untreated controls, aqueous or solvent vehicle controls (if a vehicle is used), and from the freshly sieved, pretreatment soil should be summarized in tabular form by observation date.  Means and standard deviations should be plotted for each treated sample and each control.  
      (2) NOEC.  The significance of differences between means may be established using analysis of variance and a multiple range test such as Dunnett's.  These hypothesis-testing procedures can be used to determine NOEC and LOEC values based upon the CO2 efflux, nitrification and ammonification. 
      (3) Concentration-response curve, slope, and EC50.  The EC50 for each of CO2 efflux, nitrification and ammonification should be determined.  Appropriate statistical analyses should provide a goodness-of-fit determination for the concentration-response curves.  Standard error and 95% confidence intervals for the calculated EC50 values are to be included.  The slope of the concentration-response curve, its standard error, and 95% confidence interval should also be reported.  
      (4) Statistical methods.  Statistical procedures for modeling continuous data are available and should be used.  Additional discussion about endpoints and statistical procedures is found in OCSPP 850.3000.
(g) Tabular summary of test conditions.  Table 1 lists the important conditions that should prevail during the definitive test.  Meeting these test conditions will greatly increase the likelihood that the completed test will be acceptable or valid. 
	Table 1. -- Summary of Test Conditions for Soil Microbial Community Toxicity Test
Test type
Spiked natural soil
Test duration
28 days
Substrate
Soil, < 70% sand, pH of 4 - 8, organic content 1 - 8%, CEC > 7 meq/100 g; sieved to 2 mm
Temperature 
22 °C (constant during test within +- 2 [o]C)
Lighting 
Complete darkness at all times
Test container size
Sufficient to contain 50 g soil (e.g., 0.5 pint jars)
Number of replicate containers per test treatment
10 (minimum) 
Test treatment levels
Minimum of 5 treatment levels plus appropriate controls
Test substance application method
Via aqueous stock solution, vehicle stock solution, adsorbed to the alfalfa supplement, or directly mixed into the soil. 
Measures of effect (Measurement endpoints)
EC50 and NOEC/LOEC based upon CO2 efflux, ammonification and nitrification at day 5 and 28

(h) Test validity.  This test would be considered to be unacceptable or invalid if one or more of the conditions in Table 2 occurred.  This list should not be misconstrued as limiting the reason(s) that a test could be found unacceptable or invalid.  However, except for the conditions listed in Table 2 and in OCSPP 850.3000, it is unlikely a study will be rejected when there are slight variations from guideline environmental conditions and study design unless the control organisms are significantly affected, the precision of the test is reduced, the power of a test to detect differences is reduced, and/or significant biases are introduced in defining the magnitude of effect on measurement endpoints as compared to guideline conditions.  Before departing significantly from this guideline, the investigator should contact the Agency to discuss the reason for the departure and the effect the change(s) will have on test acceptability.  In the test report, all departures from the guideline should be identified, reasons for these changes given, and their effects on test endpoints noted and discussed.
	Table 2. -- Test validity elements for the soil microbial community toxicity test
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1. All test containers were not identical and did not contain the same amount of soil from the same source.

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2. Untreated (negative controls) and appropriate solvent controls were not included in a test.  


(i) Reporting--
      (1) Background information.  Background information to be supplied in the report consists at a minimum of those background information items listed in paragraph (j)(1) of OCSPP 850.3000.
      (2) Guideline deviations.  Provide a statement of the guideline or protocol followed.  Include a description of any deviations from the test guideline or any occurrences which may have influenced the results of the test.
      (3) Test substance.  
            (i) Identification of the test substance:  common name, IUPAC and CAS names, CAS number, structural formula, source, lot or batch number, chemical state or form of the test substance, and its purity (i.e. for pesticides, the identity and concentration of active ingredient(s)).  If radiolabeled substance was used provide the radio purity and location(s) of the label.
      	(ii) Storage conditions of the test chemical or test substance and stability of the test chemical or test substance under storage conditions if stored prior to use.
      	(iii) Methods of preparation of the test substance in the range-finding and definitive test: description of test chemical introduction into the test medium (e.g., directly mixed, as an aqueous or solvent stock solution, or adsorbed onto the alfalfa supplement), the mass and volume of soil and test substance used for each treatment, how test substance was mixed into the soil.
      	(iv) For the definitive test, the number of treatments and nominal concentration at test initiation of the test substance per unit dry weight of test soil when the test substance is introduced as dissolved in water, solubilized with a vehicle, or coated on the alfalfa supplement and/or mixed directly into the soil.
      	(v) Description of stock solution preparation: the name and source of the vehicle, the nominal concentration(s) of the test substance in the vehicle, in stock solutions, or mixtures, and the vehicle concentrations used in the treatments and controls, pH of test solutions applied to the soil samples.
      (4) Soil microorganisms.  
            (i) Source of the test soil, the type of ecosystem from which it was removed, its chemical and physical characteristics (mechanical analysis), and any available geological information including soil type (classification).
            (ii) Collection method, storage, and handling of test soil prior to introduction of test substance.
		(iii) Source of alfalfa and amount added to soil.
      (5) Test system and conditions.  Description of the test system and conditions used in the definitive test and any preliminary range-finding tests.
            (i) Description of the test containers: type, size, volume, material, and conditioning method.
      	(ii) Description of incubation chamber: type and size.
      	(iii) Volume and weight of soil used in each test container.
      	(iv) Number of replicates per treatment level and control group(s).
      	(v) Frequency and methods of adding water to soil containers during the test period.
      	(vi) Description of experimental design and/or arrangement of equipment, including a diagram, if complex.
      	(vii) Methods used to determine the placement of soil containers in the incubation chamber and the assignment of test concentrations to containers to ensure randomization of exposure.
      	(viii) Test duration.
      	(ix) Methods and frequency of environmental monitoring performed during the definitive test for temperature and humidity.
      	(x) Method, type and frequency of observations made on soil microorganisms during the test.
      	(xi) For the definitive test, all analytical procedures and preservation methods should be described.  The accuracy of the method, method detection limit, and limit of quantification should be given.
	(6) Results.  
            (i) Environmental monitoring data results (temperature and humidity) in tabular form (provide raw data for measurements not made on a continuous basis), and descriptive statistics (mean, standard deviation, minimum, maximum).
      	(ii) Micrograms of CO2 evolved per gram of dry soil per hour, and micrograms of each of NH3 and NO3 present per gram of dry soil, by soil source and treated and untreated samples at day 5 and 28 in tabular form (provide raw data).  
      	(iii) For the definitive test, report the 5-day and 28-day EC50 values, with 95% confidence intervals for ammonification, nitrification, and CO2 evolution.  The slopes of the concentration-response curves, associated standard errors, and the 95% confidence intervals should be reported as well.
      	(iv) Description of statistical method(s) used, including software package, for determining EC50, LOEC and NOEC, values and the concentration-response model parameters and the basis for the choice of method.  Provide results of any goodness-of-fit tests and determination of minimum significant differences.
(j) References.  The references in this paragraph should be consulted for additional background material on this test guideline.
      (1) Hammons, A.S. (Ed.), 1981. Methods for ecological toxicology. A critical review of laboratory multispecies tests. ORNL-5708. EPA-560/11-80-026, Office of Toxic Substances, U.S. EPA, Washington, DC and Oak Ridge National Laboratory, Oak Ridge, TN. 
