Document ID: EPA-HQ-OPPT-2009-0154-0021
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2012-05-10T04:00Z

United States
Environmental Protection Agency
   Office of Chemical Safety
   and Pollution Prevention
   (7101)
EPA 712-C-014
January 2012

Ecological Effects
Test Guidelines

OCSPP 850.3300:

Modified Activated Sludge, Respiration Inhibition 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 850.3300: Modified activated sludge, respiration inhibition 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 materials used in developing this harmonized OCSPP test guideline include the OPPT guideline under 40 CFR 795.170 (proposed in the Federal Register of May 15, 1986 (51 FR 17880)); and OECD guideline 209, Activated Sludge, Respiration Inhibition Test.  This guideline was formerly Public Draft OCSPP 850.6800 (April, 1996).
(b) Purpose.  This guideline is intended for use in developing data on the effect of chemical substances and mixtures ("test chemicals" or "test substances") subject to environmental effects test regulations.  This guideline describes procedures for measuring the effect of test substances upon the respiration rate of microorganisms found in sewage treatment plants.  The Environmental Protection Agency will use data from this test to assess the hazard a test substance may present in the environment.
(c) Definitions.  The definitions in the OCSPP 850.3000 guideline apply to this guideline.  In addition, the more specific definition in this paragraph also applies:
      Respiration rate refers to the oxygen consumption of aerobic sludge or wastewater microorganisms, generally expressed as milligrams of oxygen (O2) per liter per hour.
(d) General considerations--
      (1) Summary of the test.  The method described in this test guideline assesses the effect of a test substance on microorganisms by measuring the respiration rate under defined conditions in the presence of different concentrations of the test substance.  The method is based on that described by the Ecological and Toxicological Association of the Dyestuffs Manufacturing Industry (see paragraph (j)(1) of this guideline), in which activated sludge obtained from a sewage treatment plant is used as the microbial source.  The purpose of this test guideline is to provide a rapid screening method whereby test substances which may adversely affect aerobic microbial treatment plants can be identified and to indicate suitable non-inhibitory concentrations of test substances to be used in biodegradability tests.  The test is designed to determine the quantity of test substance required to cause a 50 percent inhibition or reduction (IC50) in respiration.  Note historically in OCSPP pesticide and industrial chemical guidelines the term ECx was used to cover both the current OCSPP 850.3000 guideline definition of ECx (concentration where x percent (x%) of the population exhibit the effect (e.g., survival)) and ICx (concentration resulting in a x% decrease or inhibition effect on an attribute of the test population (e.g., respiration)).
      (2) General test guidance.  General guidance applicable to this guideline can be found in the OCSPP 850.3000 guideline.
      (3) Range-finding test.  A range-finding test is usually conducted to establish the appropriate test substance concentrations for the definitive test.  Activated sludge is exposed to a series of widely-spaced (e.g., log interval) concentrations of test substance.  The details of the range-finding 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 goal of the definitive test is to determine the concentration-response curve and IC50 value (and the 95% confidence intervals) for microbial respiration at 3 hours.  In addition, the slope of the concentration-response curve, its associated standard error and the 95% confidence interval should be determined, if possible.  For IC50 determination, a minimum of five concentrations of the test chemical, plus appropriate controls, are tested.  Analytical confirmation of the concentration of test substance in the stock solutions may be performed as described in OCSPP 850.3000.  Elements of an acceptable definitive test are given in Table 1.
      (5) Limit test.  In some situations, it is only necessary to ascertain that the IC50 is above a certain limit.  In a limit test, activated sludge is exposed to a single "limit concentration", with the appropriate controls.  The multiple-definitive test may be waived if the limit concentration has not caused greater than 50% reduction in respiration.  For most chemicals, 1000 milligrams per liter (mg/L) (based upon 100% active ingredient for pesticides), or the limits of water solubility or dispersion, are considered appropriate as the limit concentration.  Except for the number of test concentrations and endpoint determinations, an acceptable limit test follows the same test procedures and is the same duration as the multi-concentration definitive test (Table 1).
(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.  The OCSPP 850.3000 guideline lists the type of information that should be known about the test substance before testing, and discusses methods for preparation of test solutions.
      (2) Test duration.  The respiration rate of the activated sludge is typically measured at 30 minutes and at 3 hours after exposure to the test substance.  However, if only one measurement is to be done, then the 3 hour measurement should be done.
      (3) Test species--
            (i) Activated sludge.  Activated sludge from a sewage treatment plant is normally used as the microbial inoculum for the test.  Where possible activated sludge should be obtained from a sewage works treating predominantly domestic sewage.  If this is not possible, the activated sludge may be obtained from sewage works treating predominantly industrial waste water but used only following deadaptation (activated sludge solids could be maintained in modified SCAS (see the OCSPP 835.3210 guideline) or Porous Pot (see the OCSPP 835.3220 guideline) systems being fed domestic sewage for at least 3 sludge retention times).  Even so, results obtained with activated sludge from works treating industrial waste waters may be atypical.  Activated sludge may contain potentially pathogenic organisms and should be handled with care.
            (ii) Microbial inoculum.  Activated sludge (collected from a sewage treatment plant) is washed, if necessary, with tap water or an isotonic solution.  After centrifuging the supernatant is decanted.  This procedure is repeated three times.  A small amount of the washed sludge is weighed and dried.  From this result calculate the amount of wet sludge to suspended in water in order to obtain an activated sludge with a mixed liquor suspended solids level of 4 grams per liter (g/L) (+ 10%).  This level gives a concentration of 1.6 g/L in the test medium if 200 milliliters (mL) of inoculum is used in a final volume of 500 mL.  If the sludge cannot be used on the day of collection, 50 mL synthetic sewage is added to each liter of the activated sludge prepared as described; this is then aerated overnight at 20 + 2 degrees Celsius ([o]C).  It is then kept aerated for use during the day.  Before use the pH is checked and buffered, if necessary to pH 6.0 to 8.0 using sodium bicarbonate solution.  Determine the mixed liquor suspended solids.  If the same batch of sludge is to be used on subsequent days (maximum 4 days), a further 50 mL of synthetic sewage feed is added at the end of each working day.
      (4) Administration of test substance.  The sludge microorganisms are exposed under static conditions to aqueous solutions of the test substance.  Each test vessel should be inoculated with 200 mL of the microbial inoculum prepared as described in paragraph (e)(3)(ii) of this guideline.
            (i) Test substance solutions.  
                  (A) At least five concentrations, spaced by a constant factor preferably not exceeding 3.2, plus appropriate control(s) are tested.  Each control group is prepared in two sets (see paragraphs (e)(4)(i)(C), (e)(5)(i)(A) and (e)(5)(i)(B) of this guideline) to bracket the start and end of treatment solution microbial preparations).  Solutions of the test substance are freshly prepared at the start of the study using a stock solution.  The preparation of test solutions is described in the OCSPP 850.3000 guideline.
                  (B) If the test substance is not sufficiently soluble to allow preparation of a concentrated stock solution in water, it should be added directly to test vessels or, alternatively, as a concentrated stock solution in an organic solvent.  Direct addition is recommended.  If an organic solvent is used, for an acceptable test the solvent should neither significantly inhibit nor contribute to respiration (i.e., solvent control respiration should be within 15% of control respiration).
                  (C) A suggested experimental procedure which may be followed for the 3-hour contact period is:
                        (1) At time "0" 16 mL of the synthetic sewage feed is made up to 300 mL with water.  Microbial inoculum (200 mL) is added and the total mixture (500 mL) poured into the first set of control vessels (first control set, C1; see paragraph (e)(6) of this guideline for number of replicates).  Aeration at 0.5 to 1 liter per minute (L/min) is commenced using a Pasteur pipet as an aeration device.
                        (2) At time "15 minutes" (15 minutes is an arbitrary, but convenient, interval) the process described in paragraph (e)(4)(i)(C)(1) of this guideline is repeated, except that 100 mL of the test substance stock solution is added to the 16 mL of synthetic sewage feed before adding water to 300 mL and microbial inoculum (200 mL) to make a volume of 500 mL.  This mixture is then poured into the first treatment level, T1, set of vessels (see paragraph (e)(6) of this guideline for number of replicates) and aerated as described in paragraph (e)(4)(i)(C)(1) of this guideline.  This process is repeated at 15-minute intervals with different volumes of the test substance stock solution to a series of treatment vessels containing different concentrations of the test substance (i.e., second treatment level (T2) through the last (T5)).  Finally, a second control set (C2) (see paragraph (e)(6) of this guideline for number of replicates) are prepared.
                        (3) If the test substance is not sufficiently soluble to allow addition by aqueous stock solution, the appropriate proportion of the 100 mL volume of test substance stock solution is replaced with water.  For example, if 10 mL of an insoluble liquid test substance or solvent containing test substance is added directly to the test vessels, 90 mL of water is added.  If insoluble solid test substance is added to the test vessel, 100 mL of water is added.
                        (4) After 3 hours the contents of the first control vessel (performed for each replicate), C1, are poured into a measuring apparatus and the respiration rate is measured over a period of up to 10 minutes; the measuring can also be carried out directly in each vessel. 
                        (5) This determination is repeated on the contents of each treatment vessel set (i.e., first treatment level (T1) through the last treatment level (T5)) at 15-minute intervals, in such a way that the contact time in each treatment vessel is three hours.  Finally, the determination on the second control set (C2) is carried out, with a resultant contact time of three hours for this control set.
                        (6) A different regime (e.g., more than one oxygen meter) should be used when measurements are to also be made after 30 minutes of contact.
                        (7) For measurement of the chemical oxygen consumption, additional vessels are prepared containing test substance, synthetic sewage feed and water, but no activated sludge.
            (iii) Abiotic oxygen uptake.  A preliminary test (or can be conducted at same time as biotic measurements) should be conducted to determine if the test substance causes measurable abiotic oxygen consumption.  Vessels are prepared as described in (e)(4)(i) of this guideline containing test substance, synthetic sewage feed and water, but no activated sludge.  Note that an inhibitor may need to be added to prevent biological oxygen consumption.  After the appropriate contact time (should be the same as that used or to be used in the limit or definitive test), measurements of oxygen consumption are made for each treatment and control vessel.
	(5) Controls--
      (i) Negative controls.  
                  (A) Every test includes two sets of negative controls (C1 and C2) consisting of the same synthetic sewage feed, conditions, procedures, and microbial inoculum from the same source, except that none of the test substance is added.  One set of the controls (C1) is prepared at the beginning of the test before any other test solutions are prepared and the other (C2) at the end after all other test solutions have been prepared.  Two sets of a vehicle (solvent) control (SC1 and SC2) are also tested if a solvent is used.  The volume of solvent added should be less than 0.1% of the total volume.
                  (B) In the suggested experimental design described in paragraph (e)(4)(i)(C) of this guideline, the first solvent control set (SC1) is prepared immediately after the first negative control set (C1) and the second solvent control set (SC2) is prepared immediately before the last negative control set (C2) is prepared.  Where the volume of solvent used is not the same between treatment levels, for a satisfactory test the maximum volume of solvent should be used to prepare the solvent control set (i.e., SC1 and SC2).
                  (C) For an acceptable definitive or limit test, control respiration rates of C1 and C2 should be within 15% of each other and SC1 and SC2 should be within 15% of each other and the negative controls (C1 and C2).
            (ii) Reference substance.  A positive control (3, 5-dichlorophenol, a known inhibitor of respiration) is also tested to determine the IC50 on each batch of activated sludge as a means of checking that the sensitivity of the sludge is not abnormal.  For the positive control, a solution of 3, 5-dichlorophenol can be conveniently prepared by dissolving 0.5 g of 3, 5-dichlorophenol in 10 mL of a 1 normal (1N) sodium hydroxide (NaOH) solution, diluting to approximately 30 mL with reagent water, adding, while stirring, 1N sulfuric acid (H2SO4 ) solution to the point of incipient precipitation (approximately 8 mL), and finally diluting the mixture to 1 L with reagent water.  The pH should then be in the range 7 to 8.  The reference substance is tested on each batch of microbial inoculum in the same way as the test substance (see paragraph (e)(4) of this guideline).  For an acceptable definitive test, the 3-hour IC50 of 3, 5-dichlorophenol should range between 5 to 30 mg/L.
      (6) Number of replicates.  At a minimum three replicates for each concentration of test substance and each negative control group set (C1 and C2), and solvent control set (SC1 and SC2), if a solvent was used, are tested.
      (7) Facilities, apparatus and supplies--
            (i) Apparatus.  Normal laboratory equipment and especially that listed in paragraphs (e)(7)(i)(A) through (e)(7)(i)(D) of this guideline is used:
                  (A) Measuring apparatus (flat-bottom flask, stirrer bar, magnetic stirrer, oxygen electrode, and recorder).  There should be no head space and the oxygen probe should fit tightly in the neck of the measuring flask; biological oxygen demand (BOD) bottles are suitable for this purpose.  
			(B) Aeration device.
			(C) pH electrode and measuring equipment.
                  (D) For measuring oxygen concentration, a polarographic oxygen electrode, connectable to a potentiometric recorder (200 milivolt range).
            (ii) Test containers.  Glass beakers, 1 L capacity, are recommended as test vessels. 
            (iii) Cleaning.  Test containers should be cleaned before each test.  See OCSPP 850.3000.
            (iv) Synthetic sewage feed.  
                  (A) A synthetic sewage feed is made by dissolving the amounts of reagent-grade substances listed in paragraphs (e)(7)(iv)(A)(1) through (e)(7)(iv)(A)(7) of this guideline in 1 L of reagent water:
				(1) 16 g peptone.
				(2) 11 g meat extract.
				(3) 3 g urea.
				(4) 0.7 g sodium chloride (NaCl).
				(5) 0.4 g calcium chloride dihydrate (CaCl2.2H2O).
				(6) 0.2 g magnesium sulfate heptahydrate (MgSO4.7H2O).
				(7) 2.8 g dipotassium hydrogen phosphate (K2HPO4).
                  (B) This synthetic sewage feed is a 100-fold concentrate of that described in the OECD Technical Report (June 11, 1976) "Proposed method for the determination of the biodegradability of surfactants used in synthetic detergents," with dipotassium hydrogen phosphate added.
      (8) Environmental conditions.  Environmental parameters during the test are maintained as specified in paragraphs (e)(8)(i) and (e)(8)(ii) of this guideline:
            (i) Temperature.  The test temperature is 20 [o]C and it should be constant within + 2 [o]C during the test.
            (ii) Aeration.  Test containers are continuously aerated with clean, oil-free air at a flow rate of 0.5 to 1 L per minute. 
      (9) Observations--
            (i) Measurement of test substance.  Analytical confirmation of the concentration of test substance in the stock solutions may be performed as described in the OCSPP 850.3000 guideline.  Validated analytical methods are used to measure the amount of test substance in a sample before beginning the test, as described in the OCSPP 850.3000 guideline.
            (ii) Environmental conditions--
                  (A) Temperature.  Temperature should be monitored; preferably continuously but at a minimum, the maximum and minimum daily temperature should be recorded.
			(B) Aeration.  The aeration rate should be checked during the study.
            (iii) Measures of effect--
                  (A) Respiration rate.  The respiration rate of an activated sludge fed with a standard amount of synthetic sewage feed is measured and recorded after a contact time of 3 hours, or contact times of both 30 minutes and 3 hours.  The respiration rate of the same activated sludge in the presence of various concentrations of the test substance under otherwise identical conditions is also measured and recorded.
                  (B) Abiotic oxygen demand.  The respiration rate of a synthetic sewage feed is measured and recorded after a contact time of 3 hours, or contact times of both 30 minutes and 3 hours.  The respiration rate of the same sewage feed in the presence of various concentrations of the test substance under otherwise identical conditions is also measured and recorded.
(f) Treatment of results--
      (1) Respiration rate.  The respiration rate is calculated from the recorder trace as milligrams of oxygen per liter per hour (O2/L/h) between approximately 6.5 mg O2/L and 2.5 mg O2/L, or over a 10 minute period when the respiration rate is low.  The portion of the respiration curve over which the respiration rate is measured should be linear.
      (2) Percent inhibition.  In order to calculate the inhibitory effect of a test substance at a particular concentration (e.g., T1), the respiration rate at a given treatment level is expressed as a percentage of the mean respiration rates of the two control sets (C1 and C2):
		1 - [2RS/(RC1 + RC2)] x 100 = percent inhibition (%I)
		where:
		RS = oxygen-consumption rate at test substance concentration (e.g., T1)
		RC1 = oxygen-consumption rate, control 1
		RC2 = oxygen-consumption rate, control 2
      (3) Concentration-response relationship.  The IC50 and its 95% confidence limits are determined for respiration rate at 3 hours (30 min also, if measured) using an appropriate statistical model to establish the concentration-response relationship for the test substance and for the reference substance.  Various statistical procedures for modeling continuous toxicity data are available and can be used.  Additional discussion about endpoints and statistical procedures is found in the OCSPP 850.3000 guideline.
      (4) Correction for abiotic oxygen demand.  If it is determined that the test substance causes measurable abiotic oxygen consumption, the microbial respiration rate should be calculated by subtracting the abiotic oxygen uptake rate from the respiration rate determined when activated sludge was present.  These adjusted respiration values would then be used to determine %I in paragraph (f)(2) of this guideline.
      (5) Interpretation of results.  The IC50 value for respiration should be regarded merely as a guide to the likely toxicity of the test substance either to activated sludge sewage treatment or to wastewater microorganisms, since the complex interactions occurring in the environment cannot be accurately simulated in a laboratory test.
(g) Tabular summary of test conditions.  Table 1 lists the important conditions that should prevail during the definitive test.  Except for the number of test concentrations and endpoint determinations, Table 1 also lists the important conditions that should prevail during a limit 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 the Modified Activated Sludge, Respiration Inhibition Test
Test duration
3 hours
Test matrix
Synthetic sewage feed
Temperature
20 [o]C (constant during test within + 2 [o]C)
Aeration
At a flow rate of 0.5 - 1 L/min.
Test vessel size
Glass beakers, 1 L, or similar
Test vessel volume
500 mL
Microbial inoculum
Washed activated sludge prepared to give a final concentration of 1.6 g/L in test medium 
Test concentrations
Minimum of 5 test concentrations plus negative and solvent (if solvent used) controls
Number of replicate test vessels per treatment
Three for each test substance treatment and three for each negative control set (C1 and C2) and vehicle control set (SC1 and SC2), if a vehicle was used.  For abiotic oxygen demand, three for each test substance treatment and three for each negative control set (C1 and C2) and vehicle control set (SC1 and SC2), if a solvent was used.
Test concentration preparation
Aqueous solution prepared by adding test substance (directly, or in a water or a vehicle) to synthetic sewage feed
Measure of Effect or Measurement Endpoint
3-h IC50 based upon respiration rate

(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 the OCSPP 850.3000 guideline, 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 any resulting effects on test endpoints noted and discussed.

	Table 2.--Test validity elements for the Modified Activated Sludge Respiration Inhibition Test
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1. Untreated (negative) controls, solvent controls (if a solvent was used) and positive controls were not included in the test.

2. All test chambers were not identical and did not contain the same amount of activated sludge and synthetic feed.

3. The control respiration rates of C1 and C2 (beginning and end of exposure period, respectively) were not within 15% of each other and the solvent control rates SC1 and SC2 (beginning and end of exposure period, respectively), if a solvent was used, were not within 15% of each other and the negative controls (C1 and C2).

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4. The 3-h IC50 of 3,5-dichlorophenol (positive control) was not in the accepted range of 5 to 30 mg/L.

(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 the OCSPP 850.3000 guideline.
      (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) Identification of the reference substance: name, source, and its purity.
	(4) Microbial inoculum.  
      (i) Source of activated sludge.
		(ii) Activated sludge collection date and method.
            (iii) Storage duration and conditions of the activated sludge, any pretreatment or addition of synthetic feed to activated sludge or deadaptation before preparation of inoculum.
            (iv) Microbial inoculum preparation methods and concentration of mixed liquor suspended solids in the inoculum.
            (v) Date microbial inoculum is prepared and age of microbial inoculum at test initiation.
      (5) Test system and conditions.  Description of the test system and conditions used in the definitive or limit test, any preliminary range-finding tests, and any reference substance tests.
            (i) Methods of preparation of the aqueous test solutions in the range-finding and definitive test, or limit test: how test substance was introduced into the test medium (e.g., directly mixed, or as an aqueous or solvent stock solution), the volume of microbial inoculum and synthetic sewage feed used for each treatment, and the volume or mass of test substance added to each treatment.
            (ii) If stock solution was used: the name and source of the vehicle, methods of stock solution preparation, nominal test substance concentrations in stock solution(s), and volume of solvent in each treatment vessel in the definitive test or limit test.
		(iii) Test duration.
            (iv) Methods and frequency of environmental monitoring performed during the definitive or limit study and any reference substance study for temperature.
            (v) For the definitive, limit, or reference substance tests, all analytical procedures 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) in tabular form (provide raw data for measurements not made on a continuous basis), and descriptive statistics (mean, standard deviation, minimum, maximum).
      	(ii) For preliminary range-finding test, if conducted, the respiration at each test substance level and in the control(s).
      	(iii) For the definitive or limit test, the respiration at each observation period for each replicate at each treatment substance level and control(s) (provide the raw data).
      	(iv) Abiotic oxygen uptake, if any, in each replicate of each test substance level and the control(s) (provide the raw data).
      	(v) For the definitive or limit test and reference substance test, the %I in respiration at each observation period at each treatment substance level.
      	(vi) For the definitive test, graph the concentration-response curves at 30 minutes (if measured) and at the end of the test.
      	(vii) For the definitive study, the slope of the concentration-response curve at 30 minutes (if measured) and the end of the test and their standard error and 95% confidence limits.
      	(viii) For the definitive and reference substance studies, calculated IC50 at end of test, and if possible, 95% confidence limits.
      	(ix) For a limit test, report the %I at the tested limit concentration.
      	(x) Description of statistical method used, including software package, for determining IC50 values and the concentration-response model parameters and the basis for the choice of method.  Provide results of any goodness-of-fit tests.
(j) References.  The references in this paragraph should be consulted for additional background material on this test guideline.
	(1) ETAD (Ecological and Toxicological Association of Dyestuffs Manufacturing Industries) Recommended Method No. 103 (also described in references in paragraphs (j)(5) and (j)(6) of this guideline).
	(2) International Organization for Standardization, 1981.  Activated sludge respiration inhibition test.  A method for assessing the inhibition of respiration of activated sludge microorganisms by test substance.  ISO/TC 147/SC 5/WG 1, N53 No. D, June 1981.  
	(3) Broecker, B. and Zahn, R., 1977.  The performance of activated sludge plants compared with the results of various bacterial toxicity tests -- A study with 3,5-dichlorphenol.  Water Research 11:165.
	(4) Brown, D.. H. Hitz and L. Schäfer, 1981.  The assessment of the possible inhibitory effect of dyestuffs on aerobic waste-water bacteria experience with a screening test.  Chemosphere 10:245-261.
	(5) Robra, K., 1976.  Bewertung toxischer Wasserinhaltsstoffe aus ihrer Inhibitorwirkung auf die Substratoxydation von Pseudomonas Stamm Berlin mit Hilfe polarographischer Sauerstoffmessungen. (Evaluation of toxic substances contained in water from their inhibitor effect on the substrate oxidation of Pseudomonas Stamm Berlin with the help of polarographischer oxygen measurements).  gwf. wasser/abwasser 117(2), 80-86.
	(6) Schefer, W., 1977.  Testing effluent components for toxic activity on biological treatments.  Textilveredlung 6:247-250.